Medicine for inhibiting drug elimination pump

ABSTRACT

A medicament for preventive and/or therapeutic treatment of a microbial infection which comprises as an active ingredient a compound represented by the following general formula (I):  
                 
 
wherein, R 1  and R 2  represent hydrogen atom, a halogen atom, hydroxyl group or the like, W 1  represents —CH═CH—, —CH 2 O—, —CH 2 CH 2 — or the like; R 3  represents hydrogen atom, a halogen atom, hydroxyl group or an amino group; R 4  represents hydrogen atom, a group of —OZ 0-4 R 5  (Z 0-4  represents an alkylene group, a fluorine-substituted alkylene group or a single bond, and R 5  represents a cyclic alkyl group, an aryl group or the like); W 2  represents a single bond or —C(R 8 )═C(R 9 )—(R 8  and R 9  represent hydrogen atom, a halogen atom, a lower alkyl group or the like, Q represents an acidic group, but W 2  and Q may together form vinylidenethiazolidinedione or an equivalent heterocyclic ring; m and n represent an integer of 0 to 2, and q represents an integer of 0 to 3.

TECHNICAL FIELD

The present invention relates to a medicament useful for preventive andtherapeutic treatment of microbial infectious diseases.

BACKGROUND ART

For preventive or therapeutic treatment of infectious diseases caused bymicroorganisms, various antibacterial agents have so far been developed,and drugs such as β-lactam antibiotics (penicillins, cephems,monobactams, carbapenems, and penems), aminoglycosides, quinolones,macrolides, tetracyclines, rifamycins, chloramphenicols, andphosphomycins have been practically used. However, with the increase ofclinically used amount of antibacterial agents, remarkable numbers ofresistant bacterial strains to these antibacterial agents have emerged,which becomes a serious problem in the treatment of infectious diseases.

Examples of problematic bacteria, which cause particularly intractableor serious infectious diseases among those caused by resistant bacteria,include Pseudomonas aeruginosa and methicillin-resistant Staphylococcusaureus (MRSA). Antibacterial agents effective against these bacteriahave been limited so far, and it is not certain whether or nottherapeutic efficacy of the currently available drugs will be expectedin the future. In particular, no drug is available at present by whichspecifically high efficacy against resistant Pseudomonas aeruginosa canbe achieved. With the increase of aged population and the popularizationof sophisticated medical technologies including human organtransplantation and anti-cancer treatments, infections frequentlyoccurring particularly in patients with reduced immunity, i.e.,so-called opportunistic infections, have become an extremely seriousproblem in the clinical field, and under the circumstances, earlydevelopments of measures against the resistant bacteria are desired.

Recently, the presence of drug efflux pumps has recognized as abacterial excretion mechanism of drugs through researches on resistanceacquiring mechanisms of resistant bacteria. In earlier researches, apump that specifically excretes a tetracycline antibacterial agent frombacterial cells was identified in 1980 by the group of Levy, and thediscovery was noted as a major factor of the resistance to tetracycline(L. McMurry, Proc. Natl. Acad. Sci. U.S.A., 77, 3974, 1980).Furthermore, based on recent researches, the presence ofmultidrug-excreting drug efflux pumps was reported in Escherichia coli,Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus bacteria,Diplococcus pneumoniae, and Neisseria gonorrhoeae. Four multidrug effluxpumps have so far been reported as homological drug efflux pumpsderiving from Pseudomonas aeruginosa, and they have been considered as acause of low drug sensitivity inherent to Pseudomonas aeruginosa (K.Poole et al., J. Bacteriol., 175, 7363, 1993; K. Poole et al., M.Microbiol., 21, 713, 1996; T. Kohler et al., M. Microbiol., 23, 345,1997; T. Mine et al., Antimicrob. Agents Chemother., 43, 415, 1999).

The drug efflux pumps of Pseudomonas aeruginosa excrete various drugsincluding β-lactams, tetracyclines, chloramphenicols, and quinolones, towhich the drug resistance of Pseudomonas aeruginosa is attributable.

In order to overcome the problem, it will be effective to invent anantibacterial agent that has a novel structure, by which resistanceacquisition due to a drug efflux pump, one of factors of resistanceacquisition, can be avoided, or develop an agent for a combinational usewith currently available antibacterial agents that can restore theirefficacy by inhibiting functions of drug efflux pumps. As one of thelatter means, drug efflux pump inhibitors have been known (WO 01/30757).

Under the circumstances, if information as to whether or not anetiologic bacterium expresses a drug efflux pump or information as towhat kind of drug efflux pump is expressed by the microorganism can beobtained in a convenient manner, it is believed that more effectivechemotherapy will become possible. For example, when a drug efflux pumpinhibitor is used in combination which restores efficacy of an existingantibacterial agent by inhibiting the function of drug efflux pump, itis expected that judgment of appropriateness of application of a drugefflux pump inhibitor as well as selection of combinational therapyutilizing an inhibitor effective to a particular drug efflux pump and anantibacterial agent can be made by obtaining the aforementionedinformation. However, any method to conveniently obtain theaforementioned information has not been known so far. In particular, anymeans to conveniently identify a kind of drug efflux pump expressed in amicroorganism or any means to conveniently know whether or not two ormore kinds of drug efflux pumps are expressed in a microorganism has notyet been known to date.

DISCLOSURE OF THE INVENTION

Therefore, an object of the present invention is to provide a novelmedicament for the treatment of infectious diseases that improvestherapeutic efficacy of an agent against pathogenic microorganisms, inparticular, a medicament that acts on a microorganism with acquiredresistance to an antimicrobial agent, and eliminates the resistance ofthe bacteria by inhibiting a drug efflux pump so as to improvepreventive and/or therapeutic effect of the antimicrobial agent.

Another object of the present invention is to provide a method forjudging effectiveness of a drug efflux pump inhibitor against amicroorganism. A still further object of the present invention is toprovide a method for conveniently obtaining information on what kind ofdrug efflux pump is expressed by a microorganism. More specifically, theobject is to provide a method for identifying a drug efflux pumpexpressed in a microorganism, and a method for conveniently verifyingexpression of two or more kinds of drug efflux pumps in a microorganism.

In order to achieve the aforementioned first object, the inventors ofthe present invention conducted various researches to search forcompounds that eliminate resistance to an antimicrobial drug ofPseudomonas aeruginosa that has acquired the resistance. As a result,they found that the compounds represented by the following generalformula (I) or (II) had the desired activity, and thus achieved thepresent invention.

The present invention thus provides a medicament for preventive and/ortherapeutic treatment of microbial infections, which comprises as anactive ingredient a compound represented by the following generalformula (I) or a physiologically acceptable salt thereof, or a hydratethereof:

wherein,

-   -   R¹ and R² each independently represent hydrogen atom, a halogen        atom, hydroxyl group, a group of OZ₁₋₆ (the group of OZ₁₋₆        represents an alkyl group having 1-6 carbon atoms or a        fluoroalkyl group having 1-6 carbon atoms, which bonds via the        oxygen atom), a group of S(O)_(n)Z₁₋₄ (Z₁₋₄ represents an alkyl        group having 1-4 carbon atoms or a fluoroalkyl group having 1-4        carbon atoms or an alkylene group derived therefrom), a group of        N(R¹²)(R¹³) (R¹² and R¹³ each independently represent hydrogen        atom, an alkyl group having 1-4 carbon atoms or a fluoroalkyl        group having 1-4 carbon atoms), a group of Z₁₋₈ which may be        substituted (Z₁₋₈ represents an alkyl group having 1-8 carbon        atoms or a fluoroalkyl group having 1-8 carbon atoms), a 5- to        7-membered cyclic alkyl group, an aryl group, a heteroaryl        group, or a 4- to 7-membered saturated or partially saturated        heterocyclic group (the cyclic alkyl group, aryl group,        heteroaryl group and heterocyclic group may have one to three        substituents selected from the group consisting of a halogen        atom, hydroxyl group, a group of OZ₁₋₄, a group of S(O)_(n)Z₁₋₄,        a group of N(R¹²)(R¹³), a group of Z₁₋₄, carboxyl group, a group        of CO₂Z₁₋₄, group of CONH₂, a group of CONH(Z₁₋₄) and a group of        CON(Z₁₋₄)(Z₁₋₄));    -   W¹ represents a group selected from the group consisting of        —CH═CH—, —N(R¹²)CO—, —CON(R¹²)—, —CH₂O— and —CH₂CH₂— (each of        the aforementioned groups binds to the thiazole ring at the left        end);    -   R³ represents hydrogen atom, a halogen atom, hydroxyl group or        an amino group;    -   R⁴ represents a group selected from the group consisting of        hydrogen atom, a group of —OZ₀₋₄R⁵ (Z₀₋₄ represents an alkylene        group having 1-4 carbon atoms, a fluorine-substituted alkylene        group having 1-4 carbon atoms or a single bond, and R⁵        represents a 5- to 7-membered cyclic alkyl group, an aryl group,        a heteroaryl group or a 4- to 7-membered saturated or partially        saturated heterocyclic group (the cyclic alkyl group, aryl        group, heteroaryl group and heterocyclic group may have one to        three substituents selected from the group consisting of a        halogen atom, hydroxyl group, a group of OZ₁₋₄, a group of        S(O)_(n)Z₁₋₄, a group of N(R¹²)(R¹³), a group of Z₁₋₄, carboxyl        group, a group of CO₂Z₁₋₄, group of CONH₂, a group of CONH(Z₁₋₄)        and a group of CON(Z₁₋₄)(Z₁₋₄)), a group of —S(O)_(n)Z₀₋₄R⁵, a        group of —N(R⁶)(R⁷) {R⁶ and R⁷ each independently represent        hydrogen atom or Z₁₋₄, or they may bind to each other to form a        saturated or unsaturated 5- to 7-membered ring (the ring may        contain one or two hetero atoms as ring constituting atoms), and        R⁶ and R⁷ may have one to three substituents selected from the        group consisting of a halogen atom, hydroxyl group, a group of        OCON(R¹⁵)(R¹⁶), a group of CONR¹⁵)(R¹⁶), a group of        N(R¹²)CON(R¹⁵)(R¹⁶), a group of Z₁₋₄, a group of OZ₁₋₄, a group        S(O)_(n)Z₁₋₄, group of CH₂OH, a group of (CH₂)_(m)N(R¹²)(R¹³), a        group of Z₁₋₄CON(R¹⁵)(R¹⁶), a group of SO₂N(R¹²)(R¹³), a group        of OSO₂N(R¹²)(R¹³), a group of OSO₂R¹², a group of NCOZ₁₋₄R¹⁵        (in the formula, R¹⁵ and R¹⁶ independently represent hydrogen        atom, a group of Z₁₋₆R¹¹, a group of Z₁₋₄N(R¹²)(R¹³), a group of        Z₁₄OH, and a group of Z₁₋₄OZ₁₋₄), carboxyl group, cyano group, a        group of COZ₁₋₄R¹⁰, a group of CO-Z₁₋₄(R¹⁰)—N(R¹²)(R¹³) (R¹⁰ is        a substituent corresponding to a side chain on an amino acid        carbon or a group of -Z₁₋₄-R¹¹ (R¹¹ represents a substituent        which forms a quaternary salt) and a group of        a 5- or 6-membered aryl group which may be substituted and a 5-        or 6-membered unsaturated heterocyclic group which may be        substituted;    -   W² represents a single bond or —C(R⁸)═C(R⁹)— (R⁸ and R⁹ each        independently represent hydrogen atom, a halogen atom, a lower        alkyl group, an alkoxy group, cyano group, carboxyl group,        hydroxymethyl group, cyanomethyl group, vinyl group or a group        of N(R¹²)(R¹³)), Q represents an acidic group, and W² and Q may        bind together to form vinylidenethiazolidinedione in E- or        Z-configuration or an equivalent heterocyclic ring;    -   m and n each independently represent an integer of 0 to 2, and q        represents an integer of 0 to 3.

As other aspects of the present invention, provided are a medicament foreliminating resistance of a microorganism with acquired drug resistance,which comprises a compound represented by the aforementioned generalformula (I) or a physiologically acceptable salt thereof as an activeingredient; and a medicament for enhancing effect of an antimicrobialagent, which comprises a compound represented by the aforementionedgeneral formula (I) or a physiologically acceptable salt thereof as anactive ingredient. The aforementioned medicaments wherein themicroorganism is Pseudomonas aeruginosa are preferred embodiments of thepresent invention. The present invention also provides a pharmaceuticalcomposition for preventive and/or therapeutic treatment of microbialinfections, which comprises a compound represented by the aforementionedgeneral formula (I) or a physiologically acceptable salt thereoftogether with an antimicrobial agent.

As further aspects of the present invention, provided are a method forpreventive and/or therapeutic treatment of microbial infections, whichcomprises the step of administering to a mammal including human apreventively and/or therapeutically effective amount of a compoundrepresented by the aforementioned general formula (I) or aphysiologically acceptable salt thereof; a method for eliminatingresistance of a microorganism with acquired resistance to anantimicrobial agent, which comprises the step of contacting with themicroorganism an effective amount of a compound represented by theaforementioned general formula (I) or a physiologically acceptable saltthereof; a method for inhibiting acquisition of resistance to anantimicrobial agent by a microorganism, which comprises the step ofcontacting with a microorganism an effective amount of a compoundrepresented by the aforementioned general formula (I) or aphysiologically acceptable salt thereof; a method for enhancingsensitivity of a microorganism to an antimicrobial agent, whichcomprises the step of contacting with a microorganism an effectiveamount of a compound represented by the aforementioned general formula(I) or a physiologically acceptable salt thereof; and a method forimproving effect of an antimicrobial agent, which comprises the step ofadministering to a mammal including human an effective amount of acompound represented by the aforementioned general formula (I) or aphysiologically acceptable salt thereof. The compounds represented bythe aforementioned general formula (I) or physiologically acceptablesalts thereof are usually administered with one or more of antimicrobialagents simultaneously or separately, or successively. The presentinvention also provides a use of the compounds represented by theaforementioned general formula (I) or physiologically acceptable saltsthereof for the manufacture of the aforementioned medicaments.

In addition to the aforementioned inventions, the present invention alsoprovides a medicament for preventive and/or therapeutic treatment ofmicrobial infections, which comprises as an active ingredient a compoundrepresented by the following general formula (I), a physiologicallyacceptable salt thereof, or hydrates thereof

In the formula, R¹, R², R³, R⁴, W¹, W² and Q have the same meanings asthose defined above; R¹⁴ represents hydrogen atom, Z₁₋₄, Z₁₋₄R⁵ orZ₁₋₄OR⁵; and X and Y each independently represent C—H or nitrogen atom.

In order to achieve the aforementioned second object, the inventors ofthe present invention conducted various researches. As a result, theyfound that judgment of effectiveness of a combination of a drug effluxpump inhibitor and an antibacterial agent was quickly and convenientlymade by applying a method of antibiotic susceptibility test utilizingagar medium in which inhibition zones formed on the surface of themedium are observed, and that a type of drug efflux pump expressed in anetiologic bacterium was identifiable by using a combination of aparticular antibacterial agent and a drug efflux pump inhibitor, andexistence of two or more kinds of drug efflux pumps expressed in amicroorganism was verified in a simple manner. The present invention wasachieved on the basis of these findings.

The present invention thus provides a method for judging effectivenessof a drug efflux pump inhibitor against a microorganism, which comprisesthe steps of:

(A1) spreading a microorganism to be tested on a surface of an agarmedium, then providing an antibacterial agent as a spot on the surfaceof the agar medium and culturing the microorganism;

(A2) determining a growth degree of the microorganism in a region of theagar medium into which the antibacterial agent has diffused during theculture period;

(A3) determining a growth degree of the microorganism in a region of theagar medium in which the antibacterial agent that has diffused duringthe culture period and a drug efflux pump inhibitor contained in theagar medium coexist; and

(A4) judging that the drug efflux pump inhibitor is effective againstthe microorganism when the growth degree of the microorganism determinedin the step (A2) is significantly higher than the growth degree of themicroorganism determined in the step (A3).

As preferred embodiments of the aforementioned method, provided are theaforementioned method wherein the antibacterial agent is provided as aspot on the agar medium surface by means of a disk; the aforementionedmethod wherein the drug efflux pump inhibitor contained in the agarmedium is the drug efflux pump inhibitor diffused from a disk providedas a spot on the agar medium surface; the aforementioned method whereinthe drug efflux pump inhibitor contained in the agar medium is the drugefflux pump inhibitor added beforehand to the agar medium duringpreparation of the agar medium; and the aforementioned method whereinthe microorganism is Pseudomonas aeruginosa.

In another preferred embodiment, the aforementioned method furthercomprises steps of (A5) providing two or more kinds of antibacterialagents each as a spot with a space between said spots in the step (A1);(A6) determining a growth degree of the microorganism in a region of theagar medium in which the two or more kinds of antibacterial agents thathave diffused into the agar medium during culture period coexist; (A7)determining a growth degree of the microorganism in a region of the agarmedium in which the two or more kinds of antibacterial agents that havediffused during the culture period and a drug efflux pump inhibitorcontained in the agar medium coexist; and (A8) judging that the drugefflux pump inhibitor is effective against the microorganism when thegrowth degree of the microorganism determined in the step (A6) issignificantly higher than the growth degree of the microorganismdetermined in the step (A7).

As preferred embodiments of the aforementioned method, provided are theaforementioned method wherein the antibacterial agents are provided eachas a spot on the agar medium surface each by using a disk; theaforementioned method wherein the drug efflux pump inhibitor containedin the agar medium is the drug efflux pump inhibitor diffused from adisk provided as a spot on the agar medium surface; the aforementionedmethod wherein the drug efflux pump inhibitor contained in the agarmedium is the drug efflux pump inhibitor added beforehand to the agarmedium during preparation of the agar medium; and the aforementionedmethod wherein the microorganism is Pseudomonas aeruginosa.

As another aspect, the present invention provides a method foridentifying a drug efflux pump expressed in a microorganism, whichcomprises the steps of:

(B1) spreading a microorganism to be tested on a surface of an agarmedium, then providing an antibacterial agent that can be excreted by aparticular drug efflux pump as a spot on the surface of the agar mediumand culturing the microorganism;

(B2) determining a growth degree of the microorganism in a region of theagar medium into which the antibacterial agent has diffused duringculture period;

(B3) determining a growth degree of the microorganism in a region of theagar medium in which the antibacterial agent that has diffused duringthe culture period and a drug efflux pump inhibitor contained in theagar medium coexist (provided that said drug efflux pump inhibitor is aspecific inhibiter for the particular drug efflux pump); and

(B4) judging that the microorganism expresses the drug efflux pump ofthe particular type when the growth degree of the microorganism measuredin the step (B2) is significantly higher than the growth degree of themicroorganism determined in the step (B3).

As preferred embodiments of the aforementioned method, provided are theaforementioned method wherein the antibacterial agent is provided as aspot on the agar medium surface by using a disk; the aforementionedmethod wherein the drug efflux pump inhibitor contained in the agarmedium is the drug efflux pump inhibitor diffused from a disk providedas a spot on the agar medium surface; the aforementioned method whereinthe drug efflux pump inhibitor contained in the agar medium is the drugefflux pump inhibitor added beforehand to the agar medium duringpreparation of the agar medium; and the aforementioned method whereinthe microorganism is Pseudomonas aeruginosa.

As a further aspect, the present invention provides a method forverifying expression of two or more kinds of drug efflux pumps in amicroorganism, which comprises the steps of:

(C1) spreading a microorganism to be tested on a surface of an agarmedium, then providing two or more kinds of antibacterial agents(provided that each of the two or more kinds of the antibacterial agentshas different effluxing specificity by the two or more kinds of drugefflux pumps, and one of the two or more kinds of the antibacterialagents (hereinafter referred to as “Antibacterial agent (1)”) has aproperty of being excreted by only one of the two or more kinds of thedrug efflux pumps (hereinafter referred to as “Drug efflux pump (1)”),whilst the other antibacterial agent or agents have a property of beingexcreted by Drug efflux pump (1) and the other drug efflux pump orpumps);

(C2) determining a growth degree of the microorganism in a region of theagar medium into which each antibacterial agent has solely diffusedduring culture period;

(C3) determining a growth degree of the microorganism in a region of theagar medium in which each antibacterial agent that has solely diffusedduring the culture period and a drug efflux pump inhibitor contained inthe agar medium coexist (provided that the drug efflux pump inhibitor isa specific inhibiter for Drug efflux pump (1)); and

(C4) judging that the microorganism expresses Drug efflux pump (1) andone or more kinds of other drug efflux pumps when the growth degree ofthe microorganism determined in the step (C2) is significantly higherthan the growth degree of the microorganism determined in the step (C3)for Antibacterial agent (1) and the growth degree of the microorganismdetermined in the step (C2) is significantly lower than the growthdegree of the microorganism determined in the step (C3) for the otherantibacterial agent or agents.

As preferred embodiments of the aforementioned method, provided are theaforementioned method wherein the two or more kinds of antibacterialagents include a combination of a β-lactam antibiotic and a quinoloneantibacterial agent; the aforementioned method wherein one of the two ormore kinds of drug efflux pumps is a MexAB-OprM pump; and theaforementioned method wherein the microorganism is Pseudomonasaeruginosa. Also provided are the aforementioned method wherein each ofthe antibacterial agents is provided as a spot on the agar mediumsurface each by using a disk; the aforementioned method wherein the drugefflux pump inhibitor contained in the agar medium is the drug effluxpump inhibitor diffused from a disk provided as a spot on the agarmedium surface; and the aforementioned method wherein the drug effluxpump inhibitor contained in the agar medium is the drug efflux pumpinhibitor added beforehand to the agar medium during preparation of theagar medium.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows the results of determination performed by using disksaccording to the method of the present invention. In the figure, thedisk on the left side contained Aztreonam (AZT: 30 μg), the disk at thecenter contained 5% DMSO solution alone (negative control) in the caseof without efflux pump inhibitor or Compound A (50 μg) in the case ofwith efflux pump inhibitor, and the disk on the right side containedLevofloxacin (LVFX: 5 μg). Change of zone represents the change in zoneshape on the agar medium, and WB represents the result of the proteinanalysis by Western blotting method.

FIG. 2 shows the results of the measurement performed by using Etestinstead of disks. In the figure, Etest on the left side containedAztreonam (AZT: 256-0.016 μg/mL) and Etest on the right side containedLevofloxacin (LVFX: 32-0.002 μg/mL). MIC with Etest represents theminimum inhibitory concentration defined by Etest system, and WBrepresents the result of the protein analysis by Western blottingmethod.

BEST MODE FOR CARRYING OUT THE INVENTION

In the specification, the “alkyl group” means a linear, branched orcyclic alkyl group or an alkyl group consisting of a combinationthereof. The same is applied to an alkyl moiety of a substituent havingthe alkyl moiety (for example, fluoroalkyl group).

In the general formula (I), R¹ is preferably an alkyl group. Examples ofthe alkyl group include a linear or branched alkyl group having 1-8carbon atoms (for example, methyl group, ethyl group, n-propyl group,isopropyl group, n-butyl group, isobutyl group, sec-butyl group,tert-butyl group, n-pentyl group and the like), and a cyclic alkyl grouphaving 3-8 carbon atoms, preferably 3-6 carbon atoms (for example,cyclopropyl group, cyclobutyl group, cyclopentyl group, cyclohexyl groupand the like). The cyclic alkyl group may have another alkyl group or ahalogen atom, hydroxyl group or the like on the ring. R¹ is alsopreferably an aryl group, a heteroaryl group or a heterocyclic group.The aryl group may preferably has a 5- or 6-membered ring. As theheterocyclic group, a heterocyclic group having 3-8, preferably 3-6, ofring constituting atoms can be used, and the ring may be saturated orpartially saturated. As a substituent that binds to the heterocyclicgroup, for example, an alkyl group or a halogen atom is preferred.

R² is preferably hydrogen atom or a halogen atom.

W¹ is preferably a linking group having a length of two atoms, and—CH═CH—, —N(R¹²)CO—, —CON(R¹²)—, —CH₂O— and —CH₂CH₂— are more preferred.

R³ is preferably hydrogen atom, a halogen atom, amino group or hydroxylgroup.

R⁴ is preferably hydrogen atom, —OZ₀₋₄R⁵ or —N(R⁶)(R⁷).

R⁵ is preferably a 5- or 6-membered aryl group, 5- to 7-memberedalicyclic group, 4- to 7-membered saturated heterocyclic group, or 5- or6-membered unsaturated heterocyclic group, which may be substituted, andmore preferably a 4- to 7-membered saturated heterocyclic group or a 5-or 6-membered unsaturated heterocyclic group.

R⁶ and R⁷ represent hydrogen atom, an alkyl group having 1-4 carbonatoms or a fluoroalkyl group having 1-4 carbon atoms, or they bind toeach other to form a saturated or unsaturated 5- to 7-membered ring. Thering may contain one or two hetero atoms as atoms constituting the ring.R⁶ and R⁷ may have one to three substituents selected from the groupconsisting of a halogen atom, hydroxyl group, a group of OCON(R¹⁵)(R¹⁶),a group of CON(R¹⁵)(R¹⁶), a group of N(R¹²)CON(R¹⁵)(R¹⁶), a group ofZ₁₋₄, a group of OZ₁₋₄, a group S(O)_(n)Z₁₋₄, group of CH₂OH, a group of(CH₂)_(m)N(R¹²)(R¹³), a group of Z₁₋₄CON(R¹⁵)(R¹⁶), a group ofSO₂N(R¹²)(R¹³), a group of OSO₂N(R¹²)(R¹³), a group of OSO₂R¹², a groupof NCOZ₁₋₄R¹⁵ (in the formula, R¹⁵ and R¹⁶ independently representhydrogen atom, a group of Z₁₋₆R¹¹, a group of Z₁₋₄N(R¹²)(R¹³), a groupof Z₁₋₄OH, and a group of Z₁₋₄OZ₁₋₄), carboxyl group, cyano group, agroup of COZ₁₋₄R¹⁰, a group of CO-Z₁₋₄(R¹⁰)—N(R¹²)(R¹³) (R¹⁰ is asubstituent corresponding to a side chain on an amino acid carbon or agroup of -Z₁₋₄-R¹¹ (R¹¹ represents a substituent which forms aquaternary salt) and a group of

wherein R¹⁰ is a substituent on α-carbon atom of an amino acid, or agroup comprising an alkyl group or a fluoroalkyl group having 1-4 carbonatoms and a quaternary salt such as

substituted at the terminal of said alkyl or fluoroalkyl group (in thedefinitions of the aforementioned substituents, R¹² and R¹³ representhydrogen atom, an alkyl group or a fluoroalkyl group having 1-4 carbonatoms, n independently represents an integer of 0 to 2, and q representsan integer of 0 to 3). Preferred examples of R¹¹ also include aquaternary salt such

R¹⁷ represents hydrogen atom, halogen atom, hydroxyl group, carboxylgroup, carbamoyl group, N-alkylcarbamoyl group, N,N-dialkylcarbamoylgroup, dihalogenomethyl group, or trihalogenomethyl group.

More preferably, R⁶ and R⁷ form a piperidine ring or the like and saidring has a substituent such as fluorine atom, hydroxyl group, a group ofOCON(R¹⁵)(R¹⁶) and a group of CON(R¹⁵)(R¹⁶) and a group ofZ₁₋₄CON(R¹⁵)(R¹⁶) on the ring. As the ring formed by R⁶ and R⁷ bound toeach other, a piperazine ring is also preferred, and preferred examplesinclude a piperazine ring of which 4-position (nitrogen atom not boundto the pyridopyrimidine ring) is substituted with an alkyl group or afluoroalkyl group having 1-4 carbon atoms, or with other substituentsuch as COZ1₋₄R¹⁰, CO-Z₁₋₄N(R¹²)(R¹³) and

W² is preferably a single bond or a group represented by —C(R⁸)═C(R⁹)—.R⁸ and R⁹ each independently represent hydrogen atom, a halogen atom, alower alkyl group, an alkoxy group, cyano group, carboxyl group,hydroxymethyl group, cyanomethyl group, vinyl group or a group ofN(R¹²)(R¹³), more preferably hydrogen atom, a halogen atom or a loweralkyl group.

Q represents an acidic group, preferably an acidic group which iscarboxyl group, 1,2,3,4-tetrazol-5-yl group or an equivalent thereof.However, type of the acidic group is not particularly limited, and saidgroup may be any one of cyclic or non-cyclic, or a combination thereof.Examples include a lower alkoxy group, hydroxyl group, carboxyl group,N-cyanocarboxamido group, a methanesulfonylamido group having 1-3fluorine atoms, —CONH-(5-tetrazolyl) group, 5-tetrazolyl group which maybe substituted, 1,2,3-triazolyl group which may be substituted,2,4-dioxothiazolidin-5-ylidenyl group which may be substituted,4-oxo-2-thioxothiazolidin-5-ylidenyl group which may be substituted,5-oxo-4-tetrazolyl group which may be substituted,3-(5-oxo)-[1.2.4]oxadiazolidinyl group which may be substituted,2-(3,5-dioxo)-[1.2.4]oxadiazolidinyl group which may be substituted,5-(3-oxo)-[1.2.4]oxadiazolidinyl group which may be substituted,3-(5-oxo)-[1.2.4]isoxazolidyl group which may be substituted or thelike. More preferred examples include carboxyl group, 5-tetrazolyl groupwhich may be substituted, N-cyanocarboxamido group, amethanesulfonylamido group that has 1-3 fluorine atoms,—CONH-(5-tetrazolyl) group or the like.

For R¹, R², R³, R⁴, W¹, W² and Q of the compounds represented by thegeneral formula (II), those explained as for R¹, R², R³, R⁴, W¹, W² andQ in the aforementioned general formula (I) can be preferably used,respectively.

R¹⁴ is preferably an alkyl group having 1-4 carbon atoms or afluoroalkyl group having 1-4 carbon atoms.

The compounds represented by the aforementioned general formula (I) canbe produced by the following methods.

As shown in Scheme 1, Compound 3 can be obtained by heatingAminopyridine derivative 1 and malonic acid or an ester of malonic acidin a solvent such as toluene and xylene. As the ester of malonic acid,Ester 2 as a phenol ester substituted with an electron withdrawing groupsuch as a halogen atom can be used. An alkyl ester can also be used. Byformylating Compound 3 with Vilsmeier's reagent prepared from phosphorusoxychloride or oxalyl chloride and dimethylformamide (DMF), Aldehyde 4can be obtained. By subjecting Compound 4 to the Wittig reaction orHorner-Emons reaction, Acrylic acid derivative 5 can be synthesized.Where the Wittig reaction is employed, Compound 5 can be obtained byreacting an alkyloxycarbomethylenetriphenylphosphorane which may besubstituted with the aldehyde compound in an inert solvent such astetrahydrofuran, DMF or methylene chloride. Where the Horner-Emonsreaction is employed, the target compound can be synthesized by reactingthe aldehyde compound with a dialkylphosphonoacetic acid ester which maybe substituted at the 2-position in the presence of a base in an inertsolvent such as THF and DMF. Compound 7 having amino group at the2-position as a linker can be obtained by converting the hydroxyl groupat the 2-position of Compound 5 into tosyl group, mesyl group,diphenylphosphoric acid ester or the like, and then replacing theresulting group with an amine. The ester group of the acrylic acidmoiety can be hydrolyzed in the final step to obtain Compound I-A. Asthe ester group, a lower alkyl ester such as those of methyl group andethyl group, tert-butyl ester, benzyl ester, allyl ester and so forthcan be used, and they can be hydrolyzed by hydrolysis under an alkalineor acidic condition, catalytic reduction or a method by using a metalcatalyst such as palladium.

Further, Compound I-A can also be synthesized by the method shown inScheme 2. Compound 9 can be obtained by converting the hydroxyl group atthe 2-position of Compound 3 into tosyl group, mesyl group,diphenylphosphoric acid ester or the like and then replacing theresulting group with an amine. The resulting product can be formylatedwith Vilsmeier's reagent prepared from phosphorus oxychloride or oxalylchloride and dimethylformamide to obtain Aldehyde 10. By subjectingCompound 10 to the Wittig reaction or Horner-Emons reaction, Acrylicacid derivative 7 can be synthesized. Where the Wittig reaction isemployed, Compound 7 can be obtained by reacting analkyloxycarbomethylenetriphenylphosphorane which may be substituted withthe aldehyde compound in an inert solvent such as THF, DMF, toluene ormethylene chloride. Where the Horner-Emons reaction is employed, thetarget compound can be synthesized by reacting the aldehyde compoundwith a dialkylphosphonoacetic acid ester which may be substituted at the2-position in the presence of a base in an inert solvent such as THF andDMF.

According to this synthetic scheme, various primary or secondary aminescan be reacted with Compound 6 to synthesize variety of 2-substitutedderivatives by the multiple parallel synthesis method in a liquid phase.

A compound in which a substituent is introduced at the 2-position of thepyridopyrimidine ring as a linker can be synthesized by the method shownin Scheme 3.

Compound 11 can be synthesized by reacting Compound 5 with an alkylatingagent in the presence of a base. The ester portion of the resultingCompound 11 can be hydrolyzed to obtain Compound I-B in a manner similarto that for the aforementioned compound having a nitrogen atom as alinker.

Compound I-B can also be synthesized by the method shown in Scheme 4,i.e., by alkylation of Compound 3 with an alkylating agent andsubsequent formylation, olefination and deprotection of the esterportion.

Compound 1 used for the aforementioned synthesis can be synthesized asfollows.

For example, Compound 1-A of which W portion is an amide bond can besynthesized by condensing an aminothiazole derivative, which is a knowncompound or can be synthesized by a known method, and a2-aminopyridine-4-carboxylic acid derivative of which amino group isprotected by a usual method used for a reaction of forming a peptidebond, and deprotecting the protective group of the amino group.

Compound 1-B of which W portion is a double bond can be synthesized bycondensing 2-Methylthiazole derivative 17, which is a known compound orcan be synthesized by a known method, and 2-Aminopyridine-4-carbaldehydederivative 18 of which amino group is protected under a condition forthe Knoevenagel reaction, and removing the protective group of the aminogroup. As for the condition of the Knoevenagel reaction, the reactioncan also be performed by heating in acetic anhydride or in the presenceof a base such as piperidine and piperazine and in the co-presence of anacid such as acetic acid.

An anion that can be obtained by treating 2-Amino-4-methylpyridinederivative 20 of which amino group is protected with a strong base suchas n-BuLi and an aldehyde are reacted, and then the resulting hydroxylgroup can be subjected to tosylation, mesylation or the like, or theresulting product is converted into a halogenated compound such as bychlorination or bromination. The resulting product is then subjected toelimination reaction using a base such as DBU to obtain Doublebond-containing derivative 23. By removing its amino protective group,Compound 1-C can be synthesized.

Compound 1-D of which W portion is an ether bond can be synthesized bycondensing Thiazolemethyl halide 24, which is a known compound or can besynthesized by a known method, and 4-Hydroxypyridine-2-carboxylic acidester 25 in the presence of a base, hydrolyzing the ester portion of theresulting Compound 26, and then converting the produced carboxylic acidinto Amine derivative 28 by employing the Curtius rearrangement or thelike. Amino compound 1-D can also be obtained by first converting thecarboxylic acid into a condensation product with hydrazine, thenconverting the resulting product into an acid azide with a nitrous acidsalt or a derivative thereof, further subjecting the resulting productto a rearrangement reaction and, if required, removing the protectivegroup of the amino group.

Compound 1-E, where W portion is ethylene, can be synthesized byreacting an anion, obtained by treating 2-Amino-4-methylpyridinederivative 20 of which amino group is protected with a strong base suchas n-BuLi, with Thiazolemethyl halide 24 to obtain a condensationproduct and then removing the protective group of the amino group.

As shown in Scheme 10, Compound 1-E can also be synthesized by reactingan anion, obtained by treating 2-Amino-4-methylpyridine derivative 20 ofwhich amino group is protected with a strong base such as n-BuLi, withHalogenoacetic acid derivative 30 to obtain a condensation product,converting the ester portion into an amide, then converting theresulting product into a thioamide compound by using Lawesson's reagent,diphosphorous pentasulfide or the like and condensing the product with ahaloketone compound.

A compound of which W portion is a triple bond can be synthesized by thesynthetic methods described in WO9633181 and WO961024.

The compound represented by the general formula (II) can be synthesizedby the method shown in Scheme 11.

Amide derivative 35 can be obtained by convertingN-Alkyl-quinolonecarboxylic acid derivative 34 described inPCT/JP00/07565 into a mixed acid anhydride, and then reacting theresulting product with ammonia or an ethylamine having anelectron-withdrawing group (EWG) such as cyanoethylamine and3-aminopropanoic acid ester. The alkylamide derivative can be convertedinto Compound 36 by treatment with sodium azide andtrifluoromethanesulfonic acid anhydride in an acetonitrile solvent toform a tetrazole ring. Then, the product can be converted into Tetrazolecompound II-A by treatment with DUB in an inert solvent such asmethylene chloride or treatment with a base such as sodium methoxide inan alcohol.

Further, Tetrazole compound II-A can similarly be obtained by subjectingCarbamoyl derivative 35 to dehydration reaction to obtain Cyanoderivative 37 and treating the resulting compound with sodium azide andaluminum chloride in dimethylformamide. Similarly, Tetrazole compoundII-A can be produced by subjecting Carboxylic acid 34 having acinnolin-4-one or naphthylidin-4-one structure to the same treatment.

As shown in Scheme 12, where a derivative having a cinnolin-4-onestructure is desired, Compound 38, which is a known compound or can beeasily derived from a known compound, can be converted into a diazocompound by treating the amino group with sodium nitrite, and theresulting product can be reacted with a malonic acid ester to obtainCompound 39 (R⁵=H). An electrophilic regent such as an alkyl halide canbe reacted with the nitrogen atom of the resulting Compound 39 (R⁵=H) byusing a base such as sodium hydride and potassium carbonate in a solventsuch as DMF and THF, and then the product can be heated in a solventsuch as Dowtherm A and PPA and treated under an ordinary hydrolysiscondition to obtain Carboxylic acid 41. The Carboxylic acid 41 obtainedcan be subjected to a treatment similar to the aforementioned treatment(Scheme 11) to produce Tetrazole compound II-B.

As shown in Scheme 13, when W portion is —CH₂O—, Compound 42 can beconverted into an alkoxide by treatment with a strong base such assodium hydride in DMF or THF, and the alkoxide can be reacted withCompound 43, which is a known compound or can be synthesized by a knownmethod (Japanese Patent Un-examined Publication (Kokai) No. 57-144264 or60-197686), to obtain Compound 44. Subsequently, Compound 44 can beconverted into a carboxylic acid by hydrolysis and subjected to atreatment similar to the aforementioned treatment (Scheme 11) to produceTetrazole compound II-C.

As for the aforementioned production of cinnolin-4-one derivative, amethod wherein 3-chloro-4-fluoroaniline is used as a starting materialis compared with a method wherein 3,4-difluoroaniline is used,3,4-difluoroaniline will give much higher yield ofcinnolin-4-one-3-carboxylic acid as for the thermal cyclization reactionperformed in a solvent such as Dowtherm A and PPA in the production ofthe known Compound 43 and selectivity of the substitution of thealkoxide shown in Scheme 13.

As shown in Scheme 14, where the compound in which W is an olefin isdesired, Compound 45, which is a known compound or can be synthesized bya known method, can be esterified and then subjected to treatment withan oxidizing agent such as selenium dioxide, and the resulting Aldehydederivative 46 and Compound 47 can be subjected to the Wittig reactionand further subjecting the adduct to addition of ammonia to obtainCarbamoyl compound 49. The resulting Carbamoyl derivative 49 can beconverted into Tetrazole compound II-D by using the same method as inScheme 11.

The synthetic intermediates and the target compounds in theaforementioned preparations can be isolated and purified by usingmethods for isolation and purification ordinarily used in the field oforganic synthetic chemistry, for example, neutralization, filtration,extraction, drying, concentration, recrystallization, variouschromatographic techniques and the like. The synthetic intermediates maybe used in subsequent reactions without purification. When a salt ofcompound of the general formula (I) or (II) is desired, a productobtained in the form of a salt may be purified without any treatment.When a product is obtained in a free form, a salt can be formed bydissolving or suspending the product in a suitable organic solvent, andthen adding an acid or base. It is also possible to convert a compoundrepresented by the general formula (I) or (II) obtained in the form of asalt into a compound in a free form, and then convert the result into anappropriate salt.

Although it is not intended to be bound by any specific theory, thecompounds represented by the general formula (I) have an activity forinhibiting drug efflux pumps of microorganisms. More specifically, thecompounds represented by the general formula (I) can act on amicroorganism with acquired resistance to an antimicrobial agent toinhibit its drug efflux pump, and eliminate the resistance of themicroorganism. In addition, the compounds represented by the generalformula (I) can act on a microorganism to inhibit a drug efflux pump,thereby suppress the acquisition of resistance to an antimicrobial agentby a microorganism. Therefore, the medicament of the present inventionthat comprises a compound represented by the general formula (I) as anactive ingredient is useful for preventive and/or therapeutic treatmentof microbial infections, generally by a combinational administrationwith an antimicrobial agent. The medicament of the present invention isextremely useful as a medicament for preventive and/or therapeutictreatment of, in particular, infectious diseases caused by amicroorganism with acquired resistance to one or more antimicrobialagents.

Methods for using the medicament of the present invention are notparticularly limited. Examples include a method of administering one ormore antimicrobial agents, and also administering the medicament of thepresent invention simultaneously, separately, or successively to enhancethe activity of the antimicrobial agent(s); and a method of preparing apharmaceutical composition comprising one or more antimicrobial agentsand the medicament of the present invention (so-called a compound drug)and the administering the composition.

Kinds of microbial infections that are applicable by the medicament ofthe present invention are not particularly limited. Bacteria aresuitable as target microorganisms. The medicament of the presentinvention can be used for various infections by microorganisms includingGram-positive or Gram-negative bacteria, aerobic or anaerobic bacteriaand the like. The medicament of the present invention can most suitablybe used for infections by Pseudomonas aeruginosa with acquiredresistance to one or more antimicrobial agents, or infections byPseudomonas aeruginosa with low sensitivity to antimicrobial agents. Themedicament of the present invention can be used for microbial infectionsof mammals including human.

Drugs having variety of structures have been known as antimicrobialagents, and various drugs are clinically used. Kinds of antimicrobialagents that can be administered in combination with the medicament ofthe present invention are not particularly limited, and examplesinclude, for example, penicillin (penam) antibiotics, cephalosporin(cephem) antibiotics, oxacephem antibiotics, penem antibiotics,carbapenem antibiotics, monobactam antibiotics, aminoglycosideantibiotics, macrolide antibiotics, chloramphenicol antibiotics,tetracycline antibiotics, glycopeptide antibiotics, phosphomycinantibiotics, lincomycin antibiotics, sulfonamide preparations,p-aminosalicylic acid preparations, isonicotinic acid hydrazidepreparations, quinolone synthetic antimicrobial agents and the like.However, antimicrobial agents are not limited to these examples. When apharmaceutical composition comprising one or more antimicrobial agentstogether with the medicament of the present invention is manufactured,the antimicrobial agents exemplified above can also be used.

As the active ingredient of the medicament of the present invention, asubstance selected from the group consisting of the compoundsrepresented by the formula (I) and pharmaceutically acceptable saltsthereof, and hydrates thereof and solvates thereof can be used. Two ormore of the substances may be used in combination. The aforementionedsubstance, per se, may be administered as the medicament of the presentinvention. Generally, however, it is desirable that the substance isadministered in the form of a pharmaceutical composition comprising oneor more of the aforementioned substances as the active ingredienttogether with one or more pharmaceutical additives. The pharmaceuticalcomposition may optionally be added with one or more of otherpharmaceutically active ingredients such as the aforementionedantimicrobial agents and β-lactamase inhibitors.

A pharmaceutical composition for the use of administration in vivo canbe readily prepared by mixing one or more of the aforementionedsubstances as the active ingredient and one or more pharmaceuticallyacceptable additives for pharmaceutical preparations according tomethods for formulation ordinarily used in the field of manufacturingpharmacy. The route of administration of the medicament of the presentinvention is not particularly limited; however, it is desirable toappropriately chose the most effective administration route forpreventive and/or therapeutic treatment of a target infectious disease.Examples of pharmaceutical compositions suitable for oral administrationinclude, for example, capsules, powders, tablets, granules, subtilizedgranules, emulsions, syrups, solutions, suspensions and the like.Examples of pharmaceutical compositions suitable for parenteraladministration include, for example, inhalants, sprays, intrarectalpreparations, injections, drip infusions, ointments, creams, transdermalpreparations, transmucosal preparations, eye drops, nasal drops, eardrops, tape preparations, patches and the like. However, the forms ofthe medicament of the present invention are not limited to theseexamples.

Among the pharmaceutical compositions suitable for oral administration,liquid preparations such as emulsions and syrups can be prepared byusing pharmaceutical additives including water; saccharides such assucrose, sorbitol, fructose; glycols such as polyethylene glycol andpropylene glycol; oils such as sesame oil, olive oil and soybean oil;antiseptics such as p-hydroxybenzoic acid esters; flavors such asstrawberry flavor and peppermint and the like. Solid preparations suchas capsules, tablets, powders and granules can be prepared by usingexcipients such as lactose, glucose, sucrose and mannitol;disintegrating agents such as starch and sodium alginate; lubricantssuch as magnesium stearate and talc; binders such as polyvinyl alcohol,hydroxypropylcellulose and gelatin; surfactants such as fatty acidesters; plasticizers such as glycerin and the like.

Among the pharmaceutical compositions suitable for parenteraladministration, liquid preparations such as injections, drip infusionsand eye drops can preferably be prepared as sterilized isotonic liquidpreparations. For example, injections can be prepared by using anaqueous medium such as a solution of sodium chloride, a solution ofglucose, or a mixture of saline and glucose solution. The intrarectalpreparations can be prepared generally in the form of suppositories byusing carriers such as cacao butter, hydrogenated fat and hydrogenatedcarboxylic acid. For the preparation of sprays, a non-irritable carriercan be used that enables fine dispersion and enhances absorption of theaforementioned substances as the active ingredient. Examples of such acarrier include lactose, glycerin and the like. Aerosols, dry powders orthe like can also be chosen as the form of preparation. However,pharmaceutical additives used for the manufacture of the medicament ofthe present invention are not limited to those mentioned above, and anyadditives available for those skilled in the art can be used.

A dose and frequency of administration of the medicament of the presentinvention are not particularly limited, and a suitable dose can bechosen depending on the type and severity of a microbial infection, thepresence or absence of an underlying disease, the age and body weight ofa patient and the like.

The first embodiment of the present invention provided from anotheraspect is a method for judging effectiveness of a drug efflux pumpinhibitor against a microorganism, which comprises the steps of:

(A1) spreading a microorganism to be tested on a surface of an agarmedium, then providing an antibacterial agent as a spot on the surfaceof the agar medium and culturing the microorganism;

(A2) determining a growth degree of the microorganism in a region of theagar medium into which the antibacterial agent has diffused duringculture period;

(A3) determining a growth degree of the microorganism in a region of theagar medium in which the antibacterial agent that has diffused duringthe culture period and a drug efflux pump inhibitor contained in theagar medium coexist; and

(A4) judging that the drug efflux pump inhibitor is effective againstthe microorganism when the growth degree of the microorganism measuredin the step (A2) is significantly higher than the growth degree of themicroorganism measured in the step (A3).

Type of the agar medium used for the method of the present invention isnot particularly limited, and any agar media commonly used for usualantibiotic susceptibility tests may be used. Examples of the agar mediuminclude, for example, an agar medium containing nutrients including acarbon source and a nitrogen source, and specific examples thereofinclude Mueller-Hinton agar medium (Difco) and the like.

As the microbial strain, strains isolated from biosamples collected frompatients with infectious diseases and cultured in an ordinary manner, aswell as type strains may be used. As the microorganism, Pseudomonasaeruginosa is preferred. Method and conditions for spreading thebacterial strain on a surface of the agar medium is not particularlylimited, and those used for antibiotic susceptibility tests and thelike, per se, may be used. For example, a bacterial strain may be spreadon a surface by using the standard method of the Japanese Society ofChemotherapy or the agar plate dilution method defined in NCCLS(National Committee for Clinical Laboratory Standards), preferably,Mueller-Hinton agar medium or the like.

Although means for providing an antibacterial agent as a spot on theagar medium is not particularly limited, a disk usually used forantibiotic susceptibility tests and the like (circular filter paperimpregnated with a medicament) can generally be used. Disks containingvarious antibacterial agents such as β-lactam antibiotics and quinoloneantibacterial agents are commercially available, andmedicament-containing disks as commercial products, per se, can be usedfor the method of the present invention. Further, antibacterial agentcontaining strips marketed as Etest (registered trademark, AB BIODISK,Sweden) are also preferred. The term “providing as a spot” used in thespecification means that an antibacterial agent is provided in an areasufficiently smaller than the surface area of the agar medium. Theantibacterial agent is preferably provided as a single spot on the agarmedium, or may also be provided as spots at two or more differentpositions. Further, two or more kinds of antibacterial agents may beprovided as spots on the same agar medium. When antibiotic-containingdisks are not commercially available, disks may be easily prepared byimpregnating an antibiotic solution prepared at a given concentrationinto filter paper having a suitable size and shape.

As the drug efflux pump inhibitor, the compounds disclosed inInternational Patent Publication WO 01/30757 can be used. The entiredisclosure of International Patent Publication WO 01/30757 isincorporated herein by reference. Besides the aforementioned compounds,the compounds mentioned in the examples of the specification can also bepreferably used. The “drug efflux pump inhibitor” referred to in thepresent specification means a medicament having an action of inhibitingthe function of a drug efflux pump of microorganism, and any medicamentsmay be used for the method of the present invention so long as theyenhance effectiveness of an antibacterial agent on the basis of theaforementioned action.

When Pseudomonas aeruginosa is used as an object of the measurement, itis desirable to select as the drug efflux pump inhibitor an inhibitorfor the MexAB-OprM pump, which is constitutively expressed byPseudomonas aeruginosa. As the medicament having an inhibitory activityagainst excretion activity of the MexAB-OprM pump, the followingcompound (hereinafter referred to as “Compound A” in the specification)or a derivative thereof is preferably used from viewpoints of potentinhibitory activity and diffusibility on the agar medium surface. ThisCompound A acts as a specific inhibitor against the MexAB-OprM pump.

In the method of the present invention, it is necessary to form a regionin which the antibacterial agent that has diffused from a disk or thelike into the agar medium and the drug efflux pump inhibitor containedin the agar medium coexist. This coexistence state may generally beattained by (i) providing the drug efflux pump inhibitor on the agarmedium surface as a spot by means of a disk or the like so that the drugefflux pump inhibitor can diffuse therefrom into the agar medium to forma region in which it exist together with the antibacterial agent thathas diffused into the agar medium (region where the diffusions overlap),or (ii) preparing an agar medium added beforehand with the drug effluxpump inhibitor and allowing the antibacterial agent to diffuse into theagar medium from a disk or the like to form a region where the bothagents coexist. In the latter embodiment, the disk can be prepared inthe same manner as those described above.

The growth degree of the microorganism in the aforementioned coexistenceregion can be usually determined by visual inspection of the surface ofthe agar medium, and an area where no microorganism grows is generallyobserved as a clear portion. As criteria for the judgment, those adoptedin antibiotic susceptibility tests according to the standard methods ofthe Japanese Society of Chemotherapy and the like can be used withoutany modification. When the growth degree of the microorganism measuredin the step (A2) is significantly higher than the growth degree of themicroorganism measured in the step (A3), the area of the inhibition zoneobserved in the step (A3) generally becomes larger than the area of theinhibition zone observed in the step (A2) (inhibition zone may sometimesnot exist). Therefore, those skilled in the art can easily observe theaforementioned state to conduct the judgment of the step (A4).

Type of the antibacterial agent used in the aforementioned method is notparticularly limited, and any antibacterial agent may be used. Examplesinclude, for example, β-lactam antibiotics (penicillins, cephems,monobactams, carbapenems, penems), aminoglycoside antibiotics, quinoloneantibacterial agents, macrolide antibiotics, tetracycline antibiotics,rifamycin antibiotics, chloramphenicol, phosphomycin and the like.However, the antibacterial agents are not limited to these examples. Inthe aforementioned method, effectiveness of combinational use of two ormore kinds of antibacterial agents and drug efflux pump inhibitor can bejudged by providing two or more kinds of antibacterial agents each as aspot with a space between the spots, determining a growth degree of themicroorganism in a region of the agar medium in which the two or morekinds of antibacterial agents that have diffused during the cultureperiod coexist, similarly determining a growth degree of themicroorganism in a region of the agar medium in which the two or morekinds of antibacterial agents and the drug efflux pump inhibitorcoexist, and comparing the results obtained.

The second embodiment of the present invention is a method foridentifying a drug efflux pump expressed in a microorganism, whichcomprises the steps of:

(B1) spreading a microorganism to be tested on a surface of an agarmedium, then providing an antibacterial agent that can be excreted by aparticular drug efflux pump as a spot on the surface of the agar mediumand culturing the microorganism;

(B2) determining a growth degree of the microorganism in a region of theagar medium into which the antibacterial agent has diffused duringculture period;

(B3) determining a growth degree of the microorganism in a region of theagar medium in which the antibacterial agent that has diffused duringthe culture period and a drug efflux pump inhibitor contained in theagar medium coexist (provided that the drug efflux pump inhibitor is aspecific inhibiter for the particular drug efflux pump); and

(B4) judging that the microorganism expresses the drug efflux pump ofthe particular type when the growth degree of the microorganism measuredin the step (B2) is significantly higher than the growth degree of themicroorganism measured in the step (B3).

The aforementioned method can be generally used for identification wherethe microorganism expresses one kind of drug efflux pump. For example,it is known that Aztreonam, which is a β-lactam antibiotic, is excretedby the MexAB-OprM pump constitutively expressed in Pseudomonasaeruginosa. When aztreonam is used as an antibacterial agent in theaforementioned method for identification where the microorganism to betested expresses the MexAB-OprM pump, the growth degree of themicroorganism measured in the step (B2) will significantly exceed thegrowth degree of the microorganism measured in the step (B3), and henceit can be judged that the microorganism expresses the MexAB-OprM pump.Method for preparation of agar medium, method for providing anantibacterial agent as a spot, method for achieving coexistence with thedrug efflux pump inhibitor and the like used in the aforementionedmethod are the same as those explained above.

In the aforementioned method, Pseudomonas aeruginosa is preferred as themicroorganism to be tested. As the antibacterial agent, for example, aβ-lactam antibiotic or the like can be used. The β-lactam antibiotic canbe suitably selected from those commercially available as therapeuticagents, and β-lactam antibiotics such as penicillin antibiotics, cephemantibiotics, monobactam antibiotics and carbapenem antibiotics can beused. More specifically, penicillin antibiotics, cephem antibiotics,monobactam antibiotics and carbapenem antibiotics such as piperacillin,piperacillin/tazobactam, carbenicillin, ceftazidime, cefepime, aztreonamand meropenem and the like can be used. When Pseudomonas aeruginosa isused as a microorganism to be tested, it is preferable to use a β-lactamantibiotic as an antibacterial agent which is more specifically excretedby the MexAB-OprM pump compared with other drug efflux pumps. Aztreonamis a particularly preferred antibacterial agent.

The third embodiment of the present invention is a method for verifyingexpression of two or more kinds of drug efflux pumps in a microorganism,which comprises the steps of:

(C1) spreading a microorganism to be tested on a surface of an agarmedium, then providing two or more kinds of antibacterial agents(provided that each of the two or more kinds of the antibacterial agentshas different effluxing properties by the two or more kinds of drugefflux pumps, and one of the two or more kinds of the antibacterialagents (hereinafter referred to as “Antibacterial agent (1)”) has aproperty of being excreted by only one of the two or more kinds of thedrug efflux pumps (hereinafter referred to as “Drug efflux pump (1)”),whilst the other antibacterial agent or agents have a property of beingexcreted by Drug efflux pump (1) and the other drug efflux pump orpumps);

(C2) determining a growth degree of the microorganism in a region of theagar medium into which each antibacterial agent has solely diffusedduring culture period;

(C3) determining a growth degree of the microorganism in a region of theagar medium in which each antibacterial agent that has solely diffusedduring the culture period and a drug efflux pump inhibitor contained inthe agar medium coexist (provided that the drug efflux pump inhibitor isa specific inhibiter for Drug efflux pump (1)); and

(C4) judging that the microorganism expresses Drug efflux pump (1) andone or more kinds of other drug efflux pumps when the growth degree ofthe microorganism determined in the step (C2) is significantly higherthan the growth degree of the microorganism determined in the step (C3)for Antibacterial agent (1) and the growth degree of the microorganismdetermined in the step (C2) is significantly lower than the growthdegree of the microorganism determined in the step (C3) for the otherantibacterial agent or agents.

As the combination of antibacterial agents used in the aforementionedmethod, a combination of two kinds of antibacterial agents is preferred,and for example, combination of a β-lactam antibiotic and a quinoloneantibacterial agent is preferred. β-Lactam antibiotics as thoseexemplified above can be used as the β-lactam antibiotic. Among them,Aztreonam can be particularly preferably used for the aforementionedmethod, which is an antibacterial agent specifically excreted only by aMexAB-OprM pump. Further, the quinolone antibacterial agent can besuitably selected from those commercially available as therapeuticagents. For example, Levofloxacin, Ofloxacin, Ciprofloxacin, Norfloxacinand the like can be used. It is known that those quinolone antibioticsare similarly excreted by the MexAB-OprM pump as well as by other Mextype drug efflux pumps (MexCD-OprJ, MexEF-OprN, MexXY and the like), andthey can be most preferably used for the aforementioned method. As thedrug efflux pump inhibitor, the aforementioned Compound A is mostpreferably used, which is a specific inhibitor of a MexAB-OprM pump.Method for preparation of agar medium, method for providing anantibacterial agent as a spot, method for achieving coexistence with thedrug efflux pump inhibitor and the like used in the aforementionedmethod are the same as those explained above.

As an embodiment of the aforementioned method, a method will bespecifically explained wherein three kinds of agents including a drugefflux pump inhibitor, β-lactam antibiotic and quinolone antibacterialagent are provided each as a spot on a surface of an agar medium spreadwith a bacterial strain that is an object of the judgment by usingdisks, for example, as a means for provision as spots. However, themethod of the present invention is not limited to the specificembodiment.

Amounts of the β-lactam antibiotic, quinolone antibacterial agent, anddrug efflux pump inhibitor provided each as a spot are not particularlylimited, and the amounts can be suitably selected consideringdiffusibility of each agent on a surface of agar medium and culture time(diffusion time) as well as strength of the inhibitory effect againstthe drug efflux pump. For example, when Aztreonam is used as theβ-lactam antibiotic, its amount provided as a spot is suitably in therange of 10-30 μg, and when Levofloxacin is used as the quinoloneantibiotic, its amount provided as a spot is suitably in the range of1-10 μg. When the aforementioned Compound A is used as the drug effluxpump inhibitor, its amount provided as a spot is suitably in the rangeof 50-100 μg. However, the amounts to be provided as spots mayoptionally be increased or decreased depending on the type of microbialstrain, which is an object of the measurement, shapes and sizes ofinhibition zones formed around the β-lactam antibiotic and quinoloneantibacterial agent and the like.

Positions of the β-lactam antibiotic and quinolone antibacterial agentprovided as spots are not particularly limited. Preferred positions maygive overlap of an area where either of the β-lactam antibiotic or thequinolone antibacterial agent diffuses in the agar medium during theculture period with an area where the drug efflux pump inhibitordiffuses in the agar medium, and also give no overlap of an area wherethe β-lactam antibiotic diffuses during the culture period with an areaof the agar medium where the quinolone antibiotic diffuses during theculture period. It is generally preferred that the two antibacterialagents are bilaterally provided between which the drug efflux pumpinhibitor is provided.

Diffusion areas of the β-lactam antibiotic, quinolone antibacterialagent and drug efflux pump inhibitor generally change depending onfactors including type of each antibacterial agent, amount of agentprovided as a spot, type of medium, culture conditions (mainly culturetime) and the like. Accordingly, the diffusion areas can suitably becontrolled by appropriately controlling these factors. For example, thedistance between the β-lactam antibiotic and the drug efflux pumpinhibitor and the distance between the quinolone antibacterial agent andthe drug efflux pump inhibitor are preferably each in the range of about1-3 cm. Inhibition zone may be formed by each of the β-lactam antibioticand quinolone antibacterial agent alone as a control. For that purpose,they can be independently provided each as a spot at such positions thatthe agents diffused from the disks of the β-lactam antibiotic andquinolone antibacterial agent give no overlap.

Conditions of the culture on the agar medium on which surface theβ-lactam antibiotic, quinolone antibacterial agent and drug efflux pumpinhibitor are placed are not particularly limited, and they can besuitably selected depending on the conditions including type of themicroorganism, kind of the agar medium, diffusion area of eachantibacterial agents and the like. For example, the culture can beperformed at a temperature of 35-37° C. for 12-36 hours. During theculture, the β-lactam antibiotic, quinolone antibacterial agent and drugefflux pump inhibitor placed on the surface of agar medium diffuse inthe surface and inside the agar medium.

As an example of the method of the present invention, standards forjudging properties of a microbial strain based on information obtainedafter culture are explained below as for a test where Pseudomonasaeruginosa constitutively expressing an MexAB-OprM pump is used as anobject of the measurement, Aztreonam (specifically excreted only by theMexAB-OprM pump) as a β-lactam antibiotic and Levofloxacin (excreted bya MexAB-OprM pump and other Mex type drug efflux pumps) as a quinolonedrug are used as antibacterial agents, Compound A that is a specificinhibitor to a MexAB-OprM pump is used as the drug efflux pumpinhibitor, and disks containing each agent are provided on an agarmedium. However, the method of the present invention is not limited tothe aforementioned specific embodiment.

(1) Where Bacterial Strain is Pseudomonas aeruginosa that ExpressesMexAB-OprM Pump

This bacterial strain has resistance to both of Aztreonam as a β-lactamantibiotic and Levofloxacin as a quinolone antibiotic on the basis ofexcretion of the agents by a MexAB-OprM pump. Therefore, at a positionwhere the diffusion area of Aztreonam or Levofloxacin and that ofCompound A as a specific inhibitor for a MexAB-OprM pump give no overlap(only either of Aztreonam or Levofloxacin exists at this position), anyinhibition zone is not observed or only a small concentric inhibitionzone proximate to the disk is observed. This is because almost noinhibition or absolutely no inhibition of the growth of the cells isachieved solely by Aztreonam or Levofloxacin due to excretion of theantibacterial agents by the action of the MexAB-OprM pump.

Whilst, around Aztreonam or Levofloxacin at a position where thediffusion areas of Aztreonam or Levofloxacin and the diffusion area ofCompound A give overlap, a large inhibition zone is observed. This isbecause the drug excretion action of the MexAB-OprM pump is inhibited byCompound A as a MexAB-OprM pump inhibitor, and as a result, Aztreonam orLevofloxacin penetrates into the cells to enable inhibition of cellgrowth by the antibacterial activity of each antibacterial agent. Shapeof the inhibition zone observed may change depending on the degree ofoverlap of the diffusion area of Aztreonam or Levofloxacin and that ofCompound A. Further, size of the inhibition zone may also changedepending on the expression amount of the MexAB-OprM pump. When abacterial strain expresses a larger amount of the MexAB-OprM pump, theformation of the inhibition zone is enhanced. However, in each any case,the resulting inhibition zone can be clearly distinguished, based on thedifference in diameter of the inhibition zones, from the inhibition zoneformed around Aztreonam or Levofloxacin (inhibition zone may sometimesnot be formed) at a position where diffusion area thereof gives nooverlap with that of Compound A.

(2) Where Bacterial Strain is Pseudomonas aeruginosa CoexpressingAnother Drug Efflux Pump in Addition to MexAB-OprM Pump

In addition to the MexAB-OprM pump constitutively expressed inPseudomonas aeruginosa, MexCD-OprJ pump, MexEF-OprN pump, MexXY pump andthe like are know, for example, which are Mex drug efflux pumps.Further, bacterial strains expressing two or more kinds of pumps amongthe Mex pumps are also known. For strains with said pumps, an antibioticsusceptibility test in which only a β-lactam antibiotic or a quinoloneantibacterial agent is used and no MexAB-OprM pump inhibitor is used incombination fails to clarify expression amount of the MexAB-OprM pump orexistence of Mex pump expressed in addition to the MexAB-OprM pump.Whilst the method of the present invention will provide distinctiveresults.

When the objective Pseudomonas aeruginosa expresses another Mex pump inaddition to the MexAB-OprM pump, such a strain has resistance to both ofAztreonam and Levofloxacin by the synergistic excretion action of theMexAB-OprM pump and the other Mex pump. Therefore, at a position wherethe diffusion area of Aztreonam or Levofloxacin and that of Compound Agive no overlap (only Aztreonam or Levofloxacin exists at thisposition), no inhibition zone is observed or only a small concentricinhibition zone proximate to the disk is formed.

Further, at a position where the diffusion area of Levofloxacin and thatof Compound A give overlap, no formation of inhibition zone is observedor only a small inhibition zone is observed around Levofloxacin. This isbecause Levofloxacin cannot sufficiently penetrates into the cells dueto excretion by the Mex pump other than the MexAB-OprM pump, and as aresult, the agent becomes impossible to inhibit the growth of thebacterium.

Whilst, at a position where the diffusion area of Aztreonam and that ofCompound A give overlap, a large inhibition zone is formed around theβ-lactam antibiotic. The reason can be explained as follows. Compound Aas a MexAB-OprM pump inhibitor specifically inhibits only the MexAB-OprMpump, and the agent cannot inhibit another Mex pump if it is expressed.However, Aztreonam as a β-lactam antibiotic that is excreted only by theMexAB-OprM pump is not excreted by the Mex pump other than theMexAB-OprM pump. Therefore, when the MexAB-OprM pump is inhibited byCompound A, Aztreonam penetrates into the cells and inhibits the growthof the cells on the basis of its antibacterial activity.

The present invention was generally explained above, and the method ofthe present invention will be more specifically explained in theexamples of the present specification. Therefore, those skilled in theart can practice the method of the present invention by adding suitablemodifications or alterations to the method as required based on theabove general descriptions and specific explanations in the examples. Itshould be understood that those methods added with such modifications oralterations also fall within the scope of the present invention.

EXAMPLES

The present invention will be explained more specifically with referenceto the examples. However, the scope of the present invention is notlimited to the following examples.

Example 1(E)-3-[8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A) Methyl 3-{2-[(tert-butoxycarbonyl)amino]-4-pyridyl}propanoate

tert-Butyl N-(4-methyl-2-pyridyl)carbamate (5 g, 24.0 mmol) wasdissolved in tetrahydrofuran (120 ml), cooled to −78° C., and then addeddropwise with n-butyl lithium (40 ml, 60 mmol). Then, the reactionsolution was warmed and stirred at room temperature. The reactionmixture was stirred for 1 hour, then again cooled to −78° C., and addeddropwise with methyl bromoacetate (3.4 ml) dissolved in tetrahydrofuran(10 ml). After the reaction mixture was stirred for 30 minutes, thereaction was stopped with saturated brine and the reaction mixture wasextracted with ethyl acetate. The resulting organic layer was dried oversodium sulfate and concentrated under reduced pressure, and the residuewas purified by silica gel column chromatography (chloroform:ethylacetate=3:1→5:1) to obtain 3.95 g (59%) of the title compound as paleyellow crystals.

¹H-NMR (CDCl₃) δ: 1.53 (9H, s), 2.67 (2H, t, J=7.81Hz), 2.95 (2H, t,J=7.81Hz), 3.68 (3H, s), 6.82 (1H, dd, J=1.22, 5.13Hz), 7.84 (1H, s),8.13 (1H, d, J=5.13Hz)

(B) 3-{2-[(tert-Butoxycarbonyl)amino]-4-pyridyl}propanoic acid

Methyl 3-{2-[(tert-butoxycarbonyl)amino]-4-pyridyl}propanoate (30.65 g,0.11 mol) dissolved in methanol (200 ml) was added with 1 N aqueoussodium hydroxide (164 ml) and stirred at room temperature for 21 hours.After the solvent was evaporated under reduced pressure and the residuewas washed with diethyl ether, the resulting aqueous layer was addedwith concentrated hydrochloric acid until pH of the layer became 1. Thesolution was washed with ethyl acetate, and the resulting aqueous layerwas neutralized by further adding sodium hydroxide. The solution wasextracted with chloroform:methanol=10:1, and the resulting organic layerwas dried over sodium sulfate and concentrated under reduced pressure toobtain 11.16 g (38%) of the title compound as yellow crystals withoutpurification.

1H-NMR (CD₃OD) δ: 1.54 (9H, s), 2.67 (2H, t, J=7.59Hz), 2.95 (2H, t,J=7.59Hz), 6.90 (1H, d, J=5.14Hz), 7.80 (1H, s), 8.08 (1H, d, J=5.14Hz)

EI/MS; m/z: 267 (M⁺+1)

(C) tert-Butyl N-[4-(3-amino-3-oxopropyl)-2-pyridyl]carbamate

3-{2-[(tert-Butoxycarbonyl)amino]-4-pyridyl}propanoic acid (11.16 g,41.92 mmol) dissolved in tetrahydrofuran (200 ml) was added withtriethylamine (9 ml, 62.89 mmol), cooled with ice, and then addeddropwise with ethyl chloroformate (6 ml, 62.89 mmol). The reactionmixture was stirred for 10 minutes, and then added with aqueous ammonia(50 ml) dissolved in tetrahydrofuran (50 ml) at 0° C. The reactionmixture was stirred for 20 minutes under ice cooling, and then thesolvent was evaporated under reduced pressure. The resulting residue waswashed with water and extracted with chloroform. The resulting organiclayer was washed with saturated brine, and the organic layer was driedover sodium sulfate and concentrated under reduced pressure to obtain11.794 g (100%) of the title compound as brown crystals withoutpurification.

1H-NMR (CD₃OD) δ: 1.53 (9H, s), 2.57 (2H, t, J=8.05Hz), 2.97 (2H, t,J=8.05Hz), 5.40 (2H, br), 6.84 (1H, dd, J=1.46, 5.13Hz), 7.83 (1H, s),8.13 (1H, d, J=5.13Hz)

EI/MS; m/z: 266 (M⁺+1)

(D) tert-Butyl N-[4-(3-amino-3-thioxopropyl)-2-pyridyl]carbamate

Under argon atmosphere, tert-butylN-[4-(3-amino-3-oxopropyl)-2-pyridyl]-carbamate (11.79 g, 44.44 mmol)dissolved in tetrahydrofuran (100 ml) was added with Lawesson's reagent(9 g, 22.22 mmol) and stirred at 70-80° C. for 30 minutes. After thereaction mixture was returned to room temperature, the solvent wasevaporated under reduced pressure and the resulting residue was purifiedby silica gel column chromatography(chloroform→chloroform:methanol=20:1) to obtain 9.544 g (76%) of thetitle compound as pale yellow crystals.

¹H-NMR (CD₃OD) δ: 1.53 (9H, s), 2.89 (2H, t, J=8.30Hz), 3.09 (2H, t,J=8.30Hz), 6.94 (1H, d, J=5.13Hz), 7.75 (1H, s), 8.08 (1H, d, J=5.13Hz)

EI/MS; m/z: 282 (M⁺+1)

(E) 2-Bromo-1-cyclobutyl-1-ethanone

1-Cyclobutyl-1-ethanone (500 mg, 5.1 mmol) dissolved in methanol (5 ml)was added with bromine (0.3 ml, 5.6 mmol) and stirred at roomtemperature for 1 hour. The resulting ocher reaction mixture was addedwith water (3 ml) under ice cooling, and then gradually added withpotassium carbonate (350 mg). The mixture was extracted withdichloromethane, and the resulting organic layer was neutralized withsaturated aqueous sodium hydrogencarbonate and washed with saturatedbrine. The resulting organic layer was dried over magnesium sulfate andconcentrated under reduced pressure to obtain 867 mg (96%) of the titlecompound without purification.

¹H-NMR (CDCl₃) δ: 1.88 (1H,m), 2.00 (1H,m), 2.20-2.37 (4H,m), 3.60 (1H,qu, J=8.53Hz), 3.88 (2H, s)

(F) 8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one

tert-Butyl N-[4-(3-amino-3-thioxopropyl)-2-pyridyl]carbamate (1.38 g,4.9 mmol) dissolved in ethanol was added with2-bromo-1-cyclobutyl-1-ethanone (867 mg, 4.9 mmol) and refluxed at 100°C. for 1 hour. The resulting reaction solution was cooled to roomtemperature, then neutralized with saturated aqueous sodiumhydrogencarbonate and extracted with chloroform. The resulting organiclayer was washed with saturated brine, dried over magnesium sulfate andconcentrated under reduced pressure.

The resulting oily compound was added with dichloromethane (50 ml) andslowly added dropwise with trifluoroacetic acid (50 ml) under icecooling. Then, the reaction solution was warmed to room temperature andstirred 1 hour. The resulting solution was neutralized with saturatedsodium hydrogencarbonate and extracted with chloroform. The organiclayer was washed with saturated brine, and the resulting organic layerwas dried over magnesium sulfate and concentrated under reducedpressure.

The brown oily residue (1.37 g) was added with xylene (7 ml) andtrichlorophenyl malonate (2.7 g, 5.83 mmol) and refluxed by heating at140° C. for 1 hour, and the solvent was evaporated under reducedpressure. The resulting oily compound was purified by silica gelchromatography (ethyl acetate→chloroform:methanol=30:1→10:1→5:1) toobtain 657 mg (41% for the three steps) of the title compound as paleyellow crystals.

1H-NMR (CDCl₃) δ: 1.91 (1H,m), 2.01 (1H, qu, J=8.79Hz), 2.22 (2H, d qu,J=2.44, 8.79Hz), 2.34 (2H, tq, J=2.44, 8.79Hz), 3.39 (4H, dd, J=6.84,20.75Hz), 3.63 (1H, qu, J=8.79Hz), 5.33 (1H, s), 6.76 (1H, s), 7.11 (1H,dd, J=1.71, 7.08Hz), 7.40 (1H, s), 9.02 (1H, d, J=7.08Hz)

(G)8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-(3-hydroxypiperidino)-4H-pyrido-[1,2-a]pyrimidin-4-one

8-[2-(4-Cyclobutyl-1,3-thiazol-2:yl)ethyl]-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one(120 mg, 0.366 mmol) dissolved in tetrahydrofuran (4 ml) anddimethylformamide (1 ml) was added with 4-dimethylaminopyridine (60 mg,0.475 mmol) and p-tolunenesulfonyl chloride (77 mg, 0.402 mmol) at roomtemperature and stirred for 30 minutes. The solvent was evaporated underreduced pressure, and the residue was washed with water and extractedwith chloroform. The resulting organic layer was dried over magnesiumsulfate and concentrated under reduced pressure.

The resulting oily compound was added with dimethylformamide (3 ml) and3-hydroxypiperidine (45 mg, 0.44 mmol), and stirred at 60° C. for 40hours. The solvent was evaporated under reduced pressure, and then theresidue was purified by silica gel column chromatography(chloroform→chloroform:methanol=20:1) to obtain 103 mg (68% for the twosteps) of the title compound as oil.

1H-NMR (CDCl₃) δ: 1.56 (1H, m), 1.66 (1H, m), 1.62-2.09 (4H, m), 2.23(2H, d qu, J=2.20, 9.03Hz), 2.35 (2H, m), 3.15 (2H, t, J=7.32Hz), 3.35(2H, t, J=7.32Hz), 3.37 (1H, m), 3.48 (1H, m), 3.63 (1H, qu, J=9.03Hz),3.68 (1H, m), 3.84 (1H, m), 3.99 (1H, dd, J=2.93, 13.18Hz), 5.64 (1H,s), 6.71 (1H, dd, J=1.71, 7.32Hz), 6.76 (1H, s), 7.10 (1H, s), 8.76 (1H,d, J=7.32Hz)

EI/MS; m/z: 411 (M⁺+1)

(H)2-(3-{[1-(tert-Butyl)-1,1-dimethylsilyl]oxy}piperidino)-8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one

8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-(3-hydroxypiperidino)-4H-pyrido[1,2-a]pyrimidin-4-one(103 mg, 0.251 mmol) dissolved in dichloromethane (3 ml) was added withimidazole (51 mg, 0.753 mmol) and tert-butyldimethylsilyl chloride (57mg, 0.376 mmol) under ice cooling and stirred for 1 hour. Then, as thereaction did not progress, the reaction mixture was warmed to roomtemperature and further added with imidazole and tert-butyldimethylsilylchloride so that the reaction was completed. The reaction solution wasdiluted with chloroform, washed with water, dried over magnesium sulfateand then concentrated under reduced pressure. The resulting residue waspurified by silica gel column chromatography(chloroform→chloroform:methanol=20:1) to obtain 128 mg (97%) of thetitle compound as an orange oily substance.

1H-NMR (CDCl₃) δ: 0.87 (9H, s), 0.92 (6H, s), 1.53 (2H, m), 1.80-2.09(4H, m), 2.23 (2H, m), 2.34 (2H, m), 3.02 (2H, m), 3.14 (2H, t,J=8.04Hz), 3.35 (2H, t, J=8.04Hz), 3.64 (2H, m), 4.00 (1H, brd), 4.20(1H, brd), 5.59 (1H, s), 6.69 (1H, dd, J=1.95, 7.31Hz), 6.75 (1H, s),7.06 (1H, s), 8.76 (1H, d, J=7.31Hz)

EI/MS; m/z: 525 (M⁺+1)

(I)1-{8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-3-formyl-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl}-3-piperidyl formate

Under ice cooling, dimethylformamide (2 ml) was added with phosphorusoxychloride (34 μl, 0.366 mmol), and then the reaction solution wasadded dropwise with2-(3-{[1-(tert-butyl)-1,1-dimethylsilyl]oxy}piperidino)-8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one(128 mg, 0.244 mmol) dissolved in dimethylformamide under ice cooling.Then the reaction solution was warmed to room temperature. After 2hours, the reaction solution was further added with phosphorusoxychloride (34 μl) at room temperature and stirred at room temperaturefor 20 minutes. The solvent was evaporated under reduced pressure, andthe resulting residue was neutralized by adding saturated aqueous sodiumhydrogencarbonate and extracted with ethyl acetate and chloroform. Theorganic layer collected was washed with saturated brine, dried overmagnesium sulfate and concentrated under reduced pressure. The residuewas purified by silica gel column chromatography(chloroform→chloroform:methanol=20:1) to obtain 69 mg (61%) of the titlecompound as a yellow oily substance.

1H-NMR (CDCl₃) δ: 1.25-2.09 (6H, m), 2.22 (2H, dqu, J=2.45, 9.06Hz),2.34 (2H, m), 3.18 (2H, t, J=7.83Hz), 3.36 (2H, t, J=7.83Hz), 3.65 (3H,m), 3.82 (1H, dd, J=6.37, 13.47Hz), 3.91 (1H, dd, J=3.18, 13.47Hz), 5.07(1H, m), 6.77 (1H, dd, J=1.71, 7.34Hz), 6.77 (1H, s), 7.07 (1H, s), 7.98(1H, s), 8.72 (1H, d, J=7.34Hz), 10.08 (1H, s)

EI/MS; m/z: 467 (M⁺+1)

(J) tert-Butyl(E)-3-[8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-(3-formyloxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

1-{8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-3-formyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}-3-piperidylformate(69 mg, 0.148 mmol) dissolved in tetrahydrofuran (3 ml) was added with(tert-butoxycarbonylmethylene)triphenylphosphorane (84 mg, 0.222 mmol)and refluxed at 100° C. After 7 hours, the reaction solution was furtheradded with the phosphorane (90 mg) and further refluxed for 11 hours.Then the phosphorane (90 mg) was further added, and after 5 hours, thephosphorane (89 mg) was further added. The reaction solution wasrefluxed for 10 hours, then added with the phosphorane (90 mg) andrefluxed for 5 hours. The reaction solution was returned to roomtemperature, and the solvent was evaporated under reduced pressure. Theresidue was purified by thin layer silica gel chromatography(chloroform:methanol=30:1) to obtain 94 mg of the title compound asyellow crystals in a mixture with triphenylphosphine oxide.

1H-NMR (CDCl₃) δ: 1.51 (9H, s), 1.89-2.04 (6H, m), 2.22 (2H, m), 2.34(2H, m), 3.19 (2H, t, J=7.81Hz), 3.36 (2H, t, J=7.81Hz), 3.53-3.68 (4H,m), 3.79 (1H, dd, J=3.42, 13.67Hz), 5.13 (1H, m), 6.76 (1H, s), 6.84(1H, dd, J=1.95, 7.32Hz), 7.06 (1H, d, J=15.63Hz), 7.20 (1H, s), 7.65(1H, d, J=15.63Hz), 8.08 (1H, s), 8.85 (1H, d, J=7.32Hz)

(K) tert-Butyl(E)-3-[8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

tert-Butyl(E)-3-[8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-(3-formyloxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(95 mg, 0.168 mmol) dissolved in methanol (4 ml) was added with sodiummethoxide (4 mg, 0.074 mmol) under ice cooling and stirred for 45minutes under ice cooling. Then the reaction solution was further addedwith sodium methoxide (4 mg), and after 5 minutes, further added withsodium methoxide (10 mg). After the reaction solution was stirred for 15minutes, the reaction was stopped with saturated brine and added withchloroform for extraction. The organic layer was dried over magnesiumsulfate and concentrated under reduced pressure, and the residue waspurified by thin layer silica gel chromatography(chloroform:methanol=20:1) to obtain 76 mg (84%) of the title compoundas yellow crystals in a mixture with triphenylphosphine oxide.

¹H-NMR (CDCl₃) δ: 1.51(9H, s), 1.82-2.08 (6H, m), 2.17-2.25 (2H, m),2.32-2.36 (2H, m), 3.19 (2H, t, J=7.32Hz), 3.36 (2H, t, J=7.32Hz),3.53-3.65 (4H, m), 3.92 (1H, d, J=13.92Hz), 4.01 (1H, s), 6.77 (1H, s),6.86 (1H, dd, J=1.95, 7.32Hz), 7.03 (1H, d, J=15.63Hz), 7.19 (1H, s),7.62 (1H, d, J=15.63Hz), 8.85 (1H, d, J=7.32Hz)

EI/MS; m/z: 537 (M⁺+1)

(L)(E)-3-[8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

tert-Butyl(E)-3-[8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(76 mg, 0.142 mmol) was added with formic acid (3 ml) and stirred atroom temperature for 2 hours and 30 minutes. After the formic acid wasevaporated under reduced pressure, the residue was purified by thinlayer silica gel chromatography (chloroform:methanol=15:1) to obtain 39mg (55% for the three steps) of the title compound as yellow crystals.

1H-NMR (CDCl₃) δ: 1.25 (1H, m), 1.52 (1H, m), 1.83-2.08 (4H, m), 2.21(2H, d qu, J=2.44, 8.30Hz), 2.36 (2H, tq, J=2.93, 8.30Hz), 3.19 (2H, t,J=7.32Hz), 3.36 (2H, t, J=7.32Hz), 3.63 (4H, m), 3.88 (1H, d,J=13.43Hz), 4.02 (1H, brd), 6.76 (1H, s), 6.86 (1H, dd, J=2.71, 7.32Hz),7.06 (1H, d, J=15.63Hz), 7.19 (1H, s), 7.65 (1H, d, J=15.63Hz), 8.84(1H, d, J-7.32Hz)

EI/MS; m/z: 481 (M⁺+1)

Example 2(E)-3-{8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

(A)8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one

8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4H-pyrido[1,2-a]-pyrimidin-4-one(302 mg, 0.925 mmol) dissolved in tetrahydrofuran (14 ml) anddimethylformamide (6 ml) was added with 4-dimethylaminopyridine (150 mg,1.20 mmol) and p-tolunenesulfonyl chloride (194 mg, 1.02 mmol) andstirred at room temperature for 20 minutes.

After the solvent was evaporated under reduced pressure, the residue wasdiluted with chloroform and washed with water. The resulting organiclayer was dried over magnesium sulfate and concentrated under reducedpressure.

The residue was dissolved in dimethylformamide (2 ml), added withmorpholine (1 ml, 11.5 mmol) and stirred at 70° C. for 3 hours. Afterthe reaction solution was returned to room temperature, the solvent wasevaporated under reduced pressure and the residue was purified by silicagel column chromatography (chloroform→chloroform:methanol=20:1) toobtain 351 mg of the title compound as pale yellow crystals as asubstance containing dimethylformamide.

¹H-NMR (CDCl₃) δ: 1.92 (1H, m), 2.02 (1H, m), 2.23 (2H, m), 2.35 (2H,m), 3.16 (2H, t, J=8.32Hz), 3.35 (2H, t, J=8.32Hz), 3.66 (4H, t,J=5.38Hz), 3.70 (1H, m), 3.78 (4H, t, J=5.38Hz), 5.57 (1H, s), 6.74 (1H,dd, J=2.94, 7.09Hz), 6.76 (1H, s), 7.13 (1H, s), 8.78 (1H, d, J=7.09Hz)

EI/MS; m/z: 397 (M⁺+1)

(B)8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]-pyrimidine-3-carbaldehyde

Reactions were performed in the same manner as in Example 1, (I) byusing8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one(346 mg, 0.8726 mmol) to obtain 305 mg (82%) of the title compound asocher solid.

¹H-NMR (CDCl₃) δ: 1.92-2.96 (6H, m), 3.19 (2H, t, J=7.34Hz), 3.36 (2H,t, J=7.34Hz), 3.68 (1H, m)3.73 (4H, t, J=5.14Hz), 3.82 (4H, t,J=5.14Hz), 6.77 (1H, s), 6.78 (1H, dd, J=1.96, 7.34Hz), 7.08 (1H, s),8.74 (1H, d, J=7.34Hz), 10.01 (1H, s)

EI/MS; m/z: 425 (M⁺+1)

(C) tert-Butyl(E)-3-{8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

Reactions were performed in the same manner as in Example 1, (J) byusing8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-carbaldehyde(100 mg, 0.2356 mmol) to obtain 127 mg (100%) of the title compound as ayellow oily substance in a mixture with triphenylphosphine oxide.

¹H-NMR (CDCl₃) δ: 2.02 (1H, m), 2.20 (1H, m), 2.23 (2H, m), 2.34 (2H,m), 3.20 (2H, t, J=7.08Hz), 3.37 (2H, t, J=7.08Hz), 3.60 (4H, t,J=4.39Hz), 3.63 (1H, m), 3.83 (4H, t, J=4.39Hz), 6.76 (1H, s), 6.85 (1H,dd, J=1.71, 7.32Hz), 7.05 (1H, d, J=15.63Hz), 7.21 (1H, s), 7.64 (1H, d,J=15.63Hz), 8.86 (1H, d, J=7.32Hz)

EI/MS; m/z: 523 (M⁺+1)

(D)(E)-3-{8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoicacid

Reactions were performed in the same manner as in Example 1, (L) byusing tert-butyl(E)-3-{8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(127 mg, 0.2430 mmol) to obtain 82.7 mg (73%) of the title compound asyellow crystals.

¹H-NMR (CDCl₃) δ: 1.92 (1H, m), 2.01 (1H, m), 2.22 (2H, m), 2.34 (2H,m), 3.19 (2H, t, J=8.06Hz), 3.37 (2H, t, J=8.06Hz), 3.61 (4H, s), 3.64(1H, m), 3.81 (4H, s), 6.76 (1H, s), 6.86 (1H, dd, J=1.47, 7.32Hz), 7.08(1H, d, J=15.63Hz), 7.19 (1H, s), 7.64 (1H, d, J=15.63Hz), 8.87 (1H, d,J=7.32Hz)

EI/MS; m/z: 467 (M⁺+1).

IR (cm⁻¹): 2962, 2850, 1680, 1647, 1516, 1444

Example 3(E)-3-{8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoicacid

(A) Ethyl(E)-3-{8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoate

8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carbaldehyde(100 mg, 0.236 mmol) dissolved in toluene (2 ml) was added with(carbethoxyethylidene)triphenylphosphorane (102 mg, 0.283 mmol) andrefluxed by heating at 130° C. Then the regent was added until thereaction was completed, and 7 equivalents of the regent was finallyadded. After the reaction mixture was stirred for 4 days, the solventwas evaporated under reduced pressure, and the resulting residue waspurified by thin layer silica gel chromatography(chloroform:methanol=40:1) to obtain 443 mg of yellow crystals in amixture with triphenylphosphine oxide.

¹H-NMR CDCl₃) δ: 1.33 (3H, t, J=7.08Hz), 1.88 (3H, s), 1.90 (1H, m),2.05 (1H, m), 2.23 (2H, m), 2.35 (2H, m), 3.20 (2H, t, J=7.81Hz), 3.37(2H, t, J=7.81Hz), 3.57 (4H, t, J=4.88Hz), 3.64 (1H, m), 3.73 (4H, t,J=4.88Hz), 4.25 (2H, q, J=7.08Hz), 6.77 (1H, s), 6.82 (1H, d, J=7.33Hz),7.21 (1H, s), 7.57 (1H, s), 8.84 (1H, d, J=7.33Hz)

(B)(E)-3-{8-[2-(4-Cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoicacid

Ethyl(E)-3-{8-[2-(4-cyclobutyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoate(120 mg, 0.236 mmol) dissolved in methanol (5 ml) was added dropwisewith 1 N aqueous sodium hydroxide (2 ml) under ice cooling. The reactionsolution was stirred for 10 minutes and then warmed to room temperaturebecause the reaction did not progress. The reaction solution was stirredfor 45 minutes, then further added with 1 N aqueous sodium hydroxide (2ml), and after 1 hour, further added with 1 N aqueous sodium hydroxide(2 ml). The solvent was evaporated under reduced pressure, and theresulting sodium salt of the title compound was dissolved in1,4-dioxane, added with 4 N hydrochloric acid (6 ml) and stirred. Thenthe reaction solution was concentrated under reduced pressure, and theresidue was neutralized with saturated aqueous sodium hydrogencarbonateand extracted with chloroform and methanol. The organic layer was driedover magnesium sulfate and concentrated under reduced pressure. Theresulting residue was purified by thin layer silica gel chromatography(chloroform:methanol=30:1) to obtain 75 mg (66%) of the title compoundas yellow crystals.

¹H-NMR (CDCl₃) δ: 1.84 (3H, s), 1.91 (1H, m), 2.02 (1H, hex, J=9.06Hz),2.22 (2H, d qu, J=2.21, 9.06Hz), 2.34 (2H, q, J=8.57Hz), 3.16 (2H, t,J=8.08Hz), 3.36 (2H, t, J=8.08Hz), 3.56 (4H, s), 3.64 (1H, qu,J=8.57Hz), 3.70 (4H, s), 6.76 (1H, s), 6.80 (1H, dd, J=1.22, 7.10Hz),7.16 (1H, s), 7.57 (1H, s), 8.83 (1H, d, J=7.10Hz)

EI/MS; m/z: 481 (M⁺+1)

IR (cm⁻): 2958, 2919, 2850, 1666, 1641, 1440, 1251, 1115

Example 4(E)-3-{8-[2-(4-Ethyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) 4-[2-(4-Ethyl-1,3-thiazol-2-yl)ethyl]-2-pyridinamine

tert-Butyl N-[4-(3-amino-3-thioxopropyl)-2-pyridyl]carbamate (590 mg,2.10 mmol) dissolved in ethanol (20 ml) was added with1-bromo-2-butanone (0.23 ml, 2.10 mmol) and refluxed by heating at 100°C. The reaction solution was stirred for 1 hour and then returned toroom temperature. The solvent was evaporated under reduced pressure, andthe residue was diluted with chloroform. The mixture was washed withsaturated aqueous sodium hydrogencarbonate and dried over sodiumsulfate, and concentrated under reduced pressure.

The resulting residue was added with dichloromethane (20 ml) and addeddropwise with trifluoroacetic acid (20 ml) under ice cooling. Then thereaction solution was warmed to room temperature and stirred for 15hours. The trifluoroacetic acid was evaporated under reduced pressure,and the residue was neutralized with saturated sodium hydrogencarbonateand extracted with chloroform. The organic layer was washed withsaturated brine and the collected organic layer was dried over magnesiumsulfate and concentrated under reduced pressure. The resulting residuewithout purification gave 477 mg (98%) of the title compound as anorange oily substance.

¹H-NMR (CDCl₃) δ: 1.29 (3H, t, J=7.59Hz), 2.78 (2H, dq, J=0.98, 7.59Hz),2.98 (2H, t, J=8.33Hz), 3.25 (2H, t, J=8.33Hz), 6.37 (1H, s), 6.52 (1H,dd, J=1.23, 5.39Hz), 6.72 (1H, s), 7.93 (1H, d, J=5.39Hz)

(B)8-[2-(4-Ethyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one

4-[2-(4-Ethyl-1,3-thiazol-2-yl)ethyl]-2-pyridinamine (480 mg, 2.044mmol) dissolved in xylene (10 ml) was added with trichlorophenylmalonate (1 g, 2.160 mmol) and refluxed by heating at 140° C. Thereaction solution was stirred for 3 hours and returned to roomtemperature. The solvent was evaporated under reduced pressure and theresulting residue was purified by silica gel column chromatography(chloroform→chloroform:methanol=80:1→50:1→5:1) to obtain 135 mg (22%) oforange crystals.

¹H-NMR (CDCl₃) δ: 1.28 (3H, t, J=7.57Hz), 2.78 (2H, q, J=7.57Hz), 3.36(4H, dd, J=6.35, 17.82Hz), 5.33 (1H, s), 6.76 (1H, s), 7.09 (1H, d,J=7.08Hz), 7.39 (1H, s), 9.02 (1H, d, J=7.08Hz)

EI/MS; m/z: 302 (M⁺+1)

(C)8-[2-(4-Ethyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one

Reactions were performed in the same manner as in Example 2, (A) byusing8-[2-(4-ethyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one(135 mg, 0.4480 mmol) as a starting material to obtain 120 mg (72%) ofthe title compound as pale yellow crystals.

¹H-NMR(CDCl₃) δ: 1.29 (3H, t, J=7.57Hz), 2.78 (2H, dq, J=0.977, 7.57Hz),3.16 (2H, t, J=8.06Hz), 3.34 (2H, t, J=8.06Hz), 3.64 (4H, t, J=5.13Hz),3.77 (4H, t, J=5.13Hz), 5.57 (1H, s), 6.74 (1H, d, J=0.977Hz), 6.74 (1H,dd, J=1.953, 7.08Hz), 7.12 (1H, d, J=1.953Hz), 8.79 (1H, d, J=7.08Hz)

EI/MS; m/z: 371 (M⁺+1)

(D)8-[2-(4-Ethyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]-pyrimidine-3-carbaldehyde

Reactions were performed in the same manner as in Example 1, (I) byusing8-[2-(4-ethyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one(120 mg, 0.324 mmol) to obtain 116 mg (90%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.29 (3H, t, J=7.57Hz), 2.78 (2H, q, J=7.57Hz), 3.19(2H, t, J=7.08Hz), 3.35 (2H, t, J=7.08Hz), 3.73 (4H, t, J=4.15Hz), 3.82(4H, t, J=4.15Hz), 6.76 (1H, s), 6.78 (1H, dd, J=1.71, 7.32Hz), 7.07(1H, d, J=1.71Hz), 8.74 (1H, d, J=7.32Hz), 10.11 (1H, s)

EI/MS; m/z: 399 (M⁺+1)

(E) tert-Butyl(E)-3-{8-[2-(4-ethyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

Reactions were performed in the same manner as in Example 1, (J) byusing8-[2-(4-ethyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde(116 mg, 0.2911 mmol)to obtain the title compound as yellow crystals ina mixture with triphenylphosphine oxide.

¹H-NMR (CDCl₃) δ: 1.28 (3H, t, J=7.57Hz), 1.51 (9H, s), 2.78 (2H, q,J=7.57Hz), 3.20 (2H, t, J=7.08Hz), 3.36 (2H, t, J=7.08Hz), 3.60 (4H, t,J=4.88Hz), 3.83 (4H, t, J=4.88Hz), 6.74 (1H, s), 6.85 (1H, dd, J=1.71,7.32Hz), 7.05 (1H, d, J=15.87Hz), 7.20 (1H, s), 7.68 (1H, d, J=15.87Hz),8.87 (1H, d, J=7.32Hz)

(F)(E)-3-{8-[2-(4-Ethyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-2-propenoicacid

Reactions were performed in the same manner as in Example 1, (L) byusing tert-butyl(E)-3-{8-[2-(4-ethyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoate(144 mg, 0.290 mmol) to obtain 93 mg (73%) of the title compound asyellow crystals.

¹H-NMR (CDCl₃) δ: 1.29 (3H, t, J=7.57Hz), 2.79 (2H, q, J=7.57Hz), 3.21(2H, t, J=7.08Hz), 3.38 (2H, t, J=7.08Hz), 3.63 (4H, t, J=6.15Hz), 3.83(4H, t, J=6.15Hz), 6.75 (1H, s), 6.87 (1H, dd, J=1.47, 7.32Hz), 7.09(1H, d, J=15.63Hz), 7.21 (1H, s), 7.68 (1H, d, J=15.63Hz), 8.87 (1H, d,J=7.32Hz)

EI/MS; m/z: 441 (M⁺+1)

IR (cm⁻¹): 2964, 2919, 2850, 1681, 1517, 1444

Example 5(E)-3-{2-(3-Hydroxypiperidino)-8-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) 4-Methoxy-2-pyridinecarbonitrile

4-Methoxypyridine-N-oxide hydrate (1 g, 6.99 mmol) dissolved indichloromethane (4 ml) was added with trimethylsilyl cyanide (1 ml, 7.69mmol) and subsequently added dropwise with N,N-dimethylcarbamoylchloride (0.8 ml, 9.09 mmol) under ice cooling. The reaction solutionwas warmed to room temperature, stirred for 1 hour, and then addedfurther with trimethylsilyl cyanide (0.2 ml, 1.40 mmol). The reactionsolution was stirred for 19 hours, then added with 10% potassiumcarbonate (2 ml). The mixture was diluted with ethyl acetate, washedwith saturated brine and dried over magnesium sulfate, and thenconcentrated under reduced pressure. The resulting crystals were washedwith hexane to obtain 560 mg (60%) of the title compound as pinkcrystals without purification.

¹H-NMR (CDCl₃) δ: 3.92 (3H, s), 7.02 (1H, dd, J=2.44, 5.86Hz), 7.22 (1H,d, J=2.44Hz), 8.51 (1H, d, J=5.86Hz)

(B) 4-Hydroxy-2-pyridinecarbonitrile

4-Methoxy-2-pyridinecarbonitrile (544 mg, 4.055 mmol) was added with 47%hydrobromic acid (6 ml) and refluxed by heating at 130° C. The reactionsolution was stirred for 22 hours, returned to room temperature, andthen concentrated under reduced pressure. The residue was subjected toazeotropy with toluene and diethyl ether to remove excessive hydrobromicacid. The resulting white crystals were washed with diethyl ether toobtain 1.47 g of the title compound containing hydrogen bromide withoutpurification.

¹H-NMR (CD₃OD) δ: 7.48 (1H, dd, J=2.44, 6.83Hz), 7.81 (1H, d, J=2.44Hz),8.63 (1H, d, J=6.83Hz)

(C) Ethyl 4-hydroxy-2-pyridinecarboxylate

4-Hydroxy-2-pyridinecarbonitrile (25.48 g, 0.2121 mol) dissolved inethanol (400 ml) was added with concentrated hydrochloric acid (40 ml)and refluxed by heating at 110° C. for 6 days. The reaction solution wasreturned to room temperature, evaporated under reduced pressure andadded with chloroform:methanol=20:1. After insoluble crystals wereremoved by filtration, the resulting filtrate was concentrated underreduced pressure. The resulting residue was purified by silica gelcolumn chromatography (chloroform:methanol=20:1→10:1) to obtain 22.4 g(63% for the two steps) of the title compound as yellow crystals.

¹H-NMR (CD₃OD) δ: 1.32 (3H, t, J=7.32Hz), 4.52 (2H, q, J=7.32Hz), 7.18(1H, dd, J=2.69, 6.84Hz), 7.58 (1H, d, J=2.69Hz), 8.36 (1H, d, J=6.84Hz)

EI/MS; m/z: 168 (M⁺+1)

(D) 2-(Chloromethyl)-4-isopropyl-1,3-thiazole

(4-Isopropyl-1,3-thiazol-2-yl)methanol (10 g, 63.6011 mmol) dissolved indichloromethane (60 ml) was added with thionyl chloride (7 ml, 95.40mmol) under ice cooling, and then the reaction solution was returned toroom temperature and stirred for 20 minutes. The reaction solution wasconcentrated under reduced pressure, and the residue was subjected toazeotropy with toluene and then diluted with diethyl ether, and furtherneutralized with saturated aqueous sodium hydrogencarbonate. The organiclayer was washed with saturated brine, then dried over magnesium sulfateand concentrated under reduced pressure. The title compound was obtainedas the residue without purification (11 g, 100%).

¹H-NMR (CDCl₃) δ: 1.30 (6H, d, J=7.08Hz), 3.09 (1H, qu, J=7.08Hz), 4.83(2H, s), 6.91 (1H, s)

EI/MS; m/z: 176 (M⁺+1)

(E) Ethyl4-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-2-pyridinecarboxylate

Ethyl 4-hydroxy-2-pyridinecarboxylate (19.5 g, 0.057 mol) dissolved indimethylformamide (200 ml) was added with a solution of2-(chloromethyl)-4-isopropyl-1,3-thiazole (11 g, 0.063 mol) indimethylformamide (150 ml). The mixture was added with potassium iodide(9.5 g) and potassium carbonate (12 g) at room temperature, then heatedto 110° C. and stirred for 1 hour. The reaction solution was returned toroom temperature, and the solvent was evaporated under reduced pressure.The residue was diluted with chloroform and washed with water. Theresulting organic layer was washed with saturated brine, and the organiclayer was dried over magnesium sulfate and concentrated under reducedpressure. The residue was purified by silica gel chromatography(chloroform) to obtain 8.049 g (46%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.32 (6H, d, J=6.86Hz), 1.44 (3H, t, J=7.10Hz), 3.12(1H, qu, J=6.86Hz), 4.47 (2H, q, J=7.10Hz), 5.44 (2H, s), 6.94 (1H, s),7.09 (1H, dd, J=2.694, 5.63Hz), 7.80 (1H, d, J=2.694Hz), 8.58 (1H, d,J=5.63Hz)

EI/MS; m/z: 307 (M⁺+1)

(F) 4-[(4-Isopropyl-1,3-thiazol-2-yl)methoxy]-2-pyridinecarboxylic acid

Ethyl 4-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-2-pyridinecarboxylate(7.44 g, 24.28 mmol) dissolved in ethanol (50 ml) was added dropwisewith 1 N sodium hydroxide (27 ml, 26.70 mmol) at room temperature, andstirred at room temperature for 1 hour and 30 minutes. The solvent wasevaporated under reduced pressure, and the resulting Na salt crystalswere washed with ethyl acetate.

The crystals were added with 4 N hydrochloric acid (27 ml) and1,4-dioxane (40 ml), stirred and concentrated under reduced pressure.The residue was subjected to azeotropy with toluene, and the solvent wascompletely evaporated. The resulting crystals were filtered withchloroform:methanol=10:1, and the filtrate was concentrated underreduced pressure. The resulting pale yellow crystals were purified bysilica gel column chromatography (chloroform:methanol=20:1→10:1→5:1) toobtain 6.7 g (100%) of the title compound as white crystals.

¹H-NMR (CD₃OD) δ: 1.37 (6H, d, J=6.83Hz), 3.22 (1H, qu, J=6.83Hz), 5.96(2H, s), 7.54 (1H, s), 7.92 (1H, dd, J=2.68, 6.83Hz), 8.22 (1H, d,J=2.68Hz), 8.80 (1H, d, J=6.83Hz)

EI/MS; m/z: 279 (M⁺+1).

(G) tert-ButylN-{4-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-2-pyridyl}carbamate

4-[(4-Isopropyl-1,3-thiazol-2-yl)methoxy]-2-pyridinecarboxylic acid (50mg, 0.18 mmol) was added with toluene (12 ml), triethylamine (63 μl,0.45 mmol) and diphenyl phosphorylazide (78 μl, 0.36 mmol) and refluxedby heating at 140° C. for 7 hours. The reaction solution was returned toroom temperature, added with tert-butanol (12 ml) and refluxed again byheating at 140° C. The reaction solution was stirred for 18 hours andthen returned to room temperature, and the solvent was evaporated underreduced pressure. The residue was purified by thin layer silica gelchromatography (chloroform:methanol=40:1) to obtain 33 mg (53%) of thetitle compound.

¹H-NMR (CDCl₃) δ: 1.32 (6H, d, J=6.86Hz), 1.53 (9H, s), 3.11 (1H, qu,J=6.86Hz), 5.40 (2H, s), 6.60 (1H, dd, J=2.45, 5.88Hz), 6.91 (1H, s),7.73 (1H, s), 8.15 (1H, dd, J=2.45, 5.88Hz)

EI/MS; m/z: 350 (M⁺+1)

(H) 4-[(4-Isopropyl-1,3-thiazol-2-yl)methoxy]-2-pyridinamine

tert-ButylN-{4-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-2-pyridyl}carbamate (706mg, 2.202 mmol) dissolved in dichloromethane (20 ml) was added dropwisewith trifluoroacetic acid (20 ml) under ice cooling, then warmed to roomtemperature and stirred for 2 hours. The reaction solution wasconcentrated under reduced pressure, diluted with chloroform, thenneutralized with saturated aqueous sodium hydrogencarbonate andextracted with chloroform. The organic layer was dried over magnesiumsulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography(chloroform→chloroform:methanol=50:1→30:1→20:1) to obtain 331 mg (66%)of the title compound.

¹H-NMR (CDCl₃) δ: 1.32 (6H, d, J=6.83Hz), 3.11 (1H, qu, J=6.83Hz), 5.31(2H, s), 6.08 (1H, d, J=1.95Hz), 6.35 (1H, dd, J=1.95, 5.85Hz), 6.91(1H, s), 7.91 (1H, d, J=5.85Hz)

(I)2-Hydroxy-8-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-4H-pyrido[1,2-a]pyrimidin-4-one

4-[(4-Isopropyl-1,3-thiazol-2-yl)methoxy]-2-pyridinamine (378 mg, 1.52mmol) dissolved in xylene (15 ml) was added with trichlorophenylmalonate (750 mg, 1.62 mmol) and refluxed by heating at 140° C. for 1hour and 30 minutes. The reaction solution was returned to roomtemperature and concentrated under reduced pressure, and the residue waspurified by silica gel column chromatography(chloroform→chloroform:methanol=100:1→80:1→50:1) to obtain 307 mg (64%)of the title compound.

¹H-NMR (CDCl₃) δ: 1.33 (6H, d, J=6.84Hz), 3.15 (1H, qu, J=6.84Hz), 5.23(1H, s), 5.60 (2H, s), 6.95 (1H, dd, J=2.44, 7.57Hz), 7.01 (1H, s), 7.06(1H, d, J=2.44Hz), 8.99 (1H, d, J=7.57Hz)

EI/MS; m/z: 318 (M⁺+1)

(J)2-Hydroxy-8-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-4-oxo-4H-pyrido[1,2-a]-pyrimidine-3-carbaldehyde

Dimethylformamide (3 ml) was added with phosphorus oxychloride (130 μl,1.42 mmol) under ice cooling, and further added dropwise with2-hydroxy-8-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-4H-pyrido[1,2-a]pyrimidin-4-one(300 mg, 0.945 mmol) dissolved in dimethylformamide (6 ml) under icecooling. Then, the reaction solution was returned to room temperatureand stirred for 1 hour. The reaction was stopped with saturated aqueoussodium hydrogencarbonate, and the reaction solution was extracted withchloroform. The resulting organic layer was washed with saturated brine,dried over sodium sulfate and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography(chloroform→chloroform:methanol=50:1→30:1→10:1) to obtain 45 mg (14%) ofthe title compound.

¹H-NMR (CDCl₃) δ: 1.32 (6H, d, J=6.84Hz), 3.13 (1H, qu, J=6.84Hz), 5.50(2H, s), 6.94 (1H, d, J=6.85Hz), 6.99 (2H, s), 8.92 (1H, d, J=6.85Hz),10.13 (1H, s)

EI/MS; m/z: 346 (M⁺+1)

(K) tert-Butyl(E)-3-{2-hydroxy-8-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

2-Hydroxy-8-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carbaldehyde(45 mg, 0.13 mmol) dissolved in tetrahydrofuran (2 ml) anddimethylformamide (1 ml) was added with(tert-butoxycarbonylmethylene)triphenylphosphorane (60 mg, 0.16 mmol)and refluxed by heating at 100° C. for 2 hours. The reaction solutionwas returned to room temperature and concentrated under reducedpressure, and the residue was purified by thin layer silica gelchromatography (chloroform:methanol=20:1) to obtain 36 mg (62%) of thetitle compound.

¹H-NMR (CDCl₃) δ: 1.26 (6H, d, J=6.86Hz), 1.44 (9H, s), 3.08 (1H, qu,J=6.86Hz), 5.57 (2H, s), 6.82 (1H, d, J=15.92Hz), 6.90 (1H, d,J=7.83Hz), 6.96 (1H, s), 7.01 (1H, s), 7.69 (1H, d, J=15.92Hz), 8.95(1H, d, J=7.83Hz)

EI/MS; m/z: 444 (M⁺+1)

(L) tert-Butyl(E)-3-{2-(3-hydroxypiperidino)-8-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

Reactions were performed in the same manner as in Example 1, (G) byusing tert-butyl(E)-3-{2-hydroxy-8-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoate(26 mg, 0.059 mmol) to obtain 24 mg (78%) of the title compound asyellow oily compound.

¹H-NMR (CDCl₃) δ: 1.33 (6H, d, J=7.08Hz), 1.51 (9H, s), 1.60 (2H, m),1.83 (2H, m), 3.14 (1H, qu, J=7.08Hz), 3.56 (3H, m), 3.92 (1H, dd,J=3.91,13.67Hz), 4.02 (1H, m), 5.44 (2H, s), 6.75 (1H, dd, J=2.68,7.32Hz), 6.76 (1H, s), 6.97 (1H, s), 6.98 (1H, d, J=15.62Hz), 7.48 (1H,d, J=15.62Hz), 8.86 (1H, d, J=7.32Hz)

EI/MS; m/z: 527 (M⁺+1)

(M)(E)-3-{2-(3-Hydroxypiperidino)-8-[(4-isopropyl-1,3-thiazol-2-yl)methoxy]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

tert-Butyl(E)-3-{2-(3-hydroxypiperidino)-8-[(4-isopropyl-1,3-thiazol-2-yl)-methoxy]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(24 mg, 0.046 mmol) was added with a mixture of 4 N hydrochloric acidand dioxane (1 ml) and stirred at room temperature for 3 hours. Thereaction solution was concentrated under reduced pressure, and theresidue was purified by thin layer silica gel chromatography(chloroform:methanol=15:1) to obtain 24 mg (100%) of the title compoundas yellow crystals.

¹H-NMR (CD₃OD) δ: 1.33 (6H, d, J=7.08Hz), 1.57 (1H, m), 1.69 (1H, m),1.89 (1H, m), 2.07 (1H, m), 3.14 (1H, qu, J=7.08Hz), 3.16 (1H, m), 3.17(1H, m), 3.82 (2H, m), 4.04 (1H, d, J=9.52Hz), 5.54 (2H, s), 6.87 (1H,d, J=7.81Hz), 6.92 (1H, d, J=15.38Hz), 6.95 (1H, s), 7.18 (1H, s), 7.61(1H, d, J=15.38Hz), 8.81 (1H, d, J=7.81Hz)

EI/MS; m/z: 471 (M⁺+1)

Example 6(E)-3-{8-[2-(4-Ethyl-2-thienyl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-2-propenoicacid

(A) 4-Ethyl-2-thiophenecarbaldehyde

3-Ethylthiophene (2 g, 17.8 mmol) dissolved in diethyl ether (18 ml) wasadded with a solution of n-butyl lithium in hexane (1.5 M, 14 ml, 21.4mmol) at room temperature and refluxed by heating at 60° C. for 15minutes. The reaction solution was returned to room temperature andadded dropwise with dimethylformamide (2 ml, 23.2 mmol) dissolved indiethyl ether. After the reaction solution was stirred for 2 hours atroom temperature, the reaction was stopped with saturated aqueousammonium chloride, and the reaction solution was extracted withchloroform. The collected organic layer was washed with saturated brine,then dried over magnesium sulfate and concentrated under reducedpressure.

The residue was purified by silica gel column chromatography(hexane:ethyl acetate=20:1→10:1) to obtain 2 g (80%) of the titlecompound.

¹H-NMR (CDCl₃) δ: 1.27 (3H, t, J=7.59Hz), 2.68 (2H, q, J=7.59Hz), 7.39(1H, s), 7.63 (1H, s), 9.87 (1H, s)

(B) (4-Ethyl-2-thienyl)methanol

4-Ethyl-2-thiophenecarbaldehyde (1 g, 7.13 mmol) dissolved in methanol(7 ml) was added with sodium borohydride (135 mg, 3.57 mmol) under icecooling, stirred for 10 minutes and then further added with sodiumborohydride (150 mg, 3.96 mmol) at 0° C. After the reaction solution wasstirred under ice cooling for 30 minutes, the reaction was stopped withsaturated aqueous ammonium chloride and the reaction solution wasextracted with chloroform. The organic layer collected was dried overmagnesium sulfate and concentrated under reduced pressure to obtain 1 g(100%) of the title compound without purification.

¹H-NMR (CDCl₃) δ: 1.22 (3H, t, J=7.56Hz), 2.59 (2H, q, J=7.56Hz), 4.75(2H, s), 6.86 (2H, s)

(C) tert-ButylN-{4-[2-(3-bromo-4-ethyl-2-thienyl)ethyl]-2-pyridyl}carbamate(4-Ethyl-2-thienyl)methanol (1 g, 7.03 mmol) dissolved indichloromethane (7 ml) was added with thionyl bromide (0.8 ml, 10.55mmol) under ice cooling and then warmed to room temperature. Thereaction solution was stirred for 20 minutes and then concentrated underreduced pressure, and the residue was neutralized by adding saturatedaqueous sodium hydrogencarbonate and extracted with diethyl ether. Theorganic layer was washed with saturated brine, dried over magnesiumsulfate and concentrated under reduced pressure. The residue was used inthe subsequent reaction without purification.

tert-Butyl N-(4-methyl-2-pyridyl)carbamate (800 mg, 3.84 mmol) dissolvedin tetrahydrofuran (15 ml) was cooled to −78° C. and then added dropwisewith a solution of n-butyl lithium in hexane (1.5 M, 6.4 ml, 9.6 mmol).The reaction solution was stirred at room temperature for 1 hour, thencooled to −78° C. again and added dropwise with a solution of the aboveobtained 3-bromo-2-(bromomethyl)-4-ethylthiophene in tetrahydrofuran (14ml). After the reaction solution was stirred at −78° C. for 1 hour andthe reaction was stopped with saturated brine, the reaction solution wasextracted with ethyl acetate. The organic layer collected was dried overmagnesium sulfate and concentrated under reduced pressure. The resultingresidue was purified by silica gel column chromatography (chloroform) toobtain 1.3 g (85%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.15 (3H, t, J=7.57Hz), 1.55 (9H, s), 2.52 (2H, q,J=7.57Hz), 2.92 (2H, t, J=8.55Hz), 3.04 (2H, t, J=8.55Hz), 6.50 (1H, s),6.77 (1H, d, J=5.13Hz), 7.87 (1H, s), 8.18 (1H, d, J=5.13Hz)

EI/MS; m/z: 411 (M⁺)

(D) 4-[2-(3-Bromo-4-ethyl-2-thienyl)ethyl]-2-pyridylamine

tert-Butyl N-{4-[2-(3-bromo-4-ethyl-2-thienyl)ethyl]-2-pyridyl}carbamate(1.34 g, 4.04 mmol) dissolved in dichloromethane (40 ml) was added withtrifluoroacetic acid (40 ml) at 0° C. and stirred at 0° C. for 3 hours.The reaction solution was concentrated under reduced pressure,neutralized with saturated sodium hydrogencarbonate and extracted withchloroform, and then the organic layer was dried over magnesium sulfateand concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (chloroform:ethyl acetate=3:1→1:1→ethylacetate only) to obtain 524 mg (56%) of the title compound.

¹H-NMR (CD₃OD) δ: 1.12 (3H, t, J=7.56Hz), 2.48 (2H, q, J=7.56Hz), 2.79(2H, t, J=7.56Hz), 3.01 (2H, t, J=7.56Hz), 6.40 (1H, s), 6.46 (1H, d,J=5.36Hz), 6.53 (1H, s), 7.76 (1H, d, J=5.36Hz)

EI/MS; m/z: 310 (M⁺−1)

(E)8-[2-(3-Bromo-4-ethyl-2-thienyl)ethyl]-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one

Reactions were performed in the same manner as in Example 4, (B) byusing 4-[2-(3-bromo-4-ethyl-2-thienyl)ethyl]-2-pyridylamine as astarting material. After the reaction was completed, the reactionsolution was concentrated under reduced pressure and the crystals in theresulting turbid solution was removed by filtration. The filtrate wasconcentrated under reduced pressure, and the resulting residue waspurified by silica gel column chromatography(chloroform→chloroform:ethyl acetate=1:1→ethyl acetate→ethylacetate:methanol=50:1→30:1→10:1) to obtain 180 mg (38%) of the titlecompound.

¹H-NMR (CDCl₃) δ: 1.14 (3H, t, J=7.56Hz), 2.49 (2H, q, J=7.56Hz), 3.15(4H, s), 5.31 (1H, s), 6.53 (1H, s), 7.12 (1H, d, J=7.07Hz), 7.24 (1H,s), 8.98 (1H, d, J=7.07Hz)

(F)8-[2-(4-Ethyl-2-thienyl)ethyl]-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one

8-[2-(3-Bromo-4-ethyl-2-thienyl)ethyl-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one(96 mg, 0.25 mmol) dissolved in toluene (6 ml) was added withtributyltin hydride (75 μl) and 2,2-azobisisobutyronitrile (4 mg, 0.025mmol) and refluxed by heating at 140° C. Then 2,2-azobisisobutyronitrileand tributyltin hydride were further added until the reaction wascompleted while the progress of the reaction was monitored by LC-MS. 34mg (0.22 mmol) of 2,2-azobisisobutyronitrile and 350 μl (1.30 mmol) oftributyltin hydride were used. The reaction solution was returned toroom temperature and the reaction was stopped with an aqueous solutionof potassium fluoride. The reaction solution was extracted with ethylacetate, and the organic layer was washed with saturated brine. Then,the aqueous layer was extracted with ethyl acetate. The organic layercollected was dried over magnesium sulfate and concentrated underreduced pressure. The residue was purified by thin layer silica gelchromatography (chloroform:methanol=20:1) to obtain 119 mg of the titlecompound with contained impurities.

¹H-NMR (CDCl₃) δ: 1.28 (3H, t, J=7.32Hz), 2.56 (2H, q, J=7.32Hz), 3.04(2H, t, J=8.06Hz), 3.17 (2H, t, J=8.06Hz), 5.34 (1H, s), 5.63 (1H, s),6.74 (1H, d, J=4.88Hz), 7.04 (1H, d, J=7.08Hz), 7.35 (1H, s), 9.02 (1H,d, J=7.08Hz)

ES-MS: 301 (M⁺+1)

(G)8-[2-(4-Ethyl-2-thienyl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one

Reactions were performed in the same manner as in Example 2, (A) byusing8-[2-(4-ethyl-2-thienyl)ethyl]-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one(122 mg, 0.406 mmol) as a starting material. As a result, 122 mg of thetitle compound was obtained as a yellow oily substance in a mixture withimpurities.

¹H-NMR (CDCl₃) δ: 1.20 (3H, t, J=7.57Hz), 2.56 (2H, q, J=7.57Hz), 3.01(2H, t, J=7.32Hz), 3.16 (2H, t, J=7.32Hz), 3.66 (4H, t, J=4.88Hz), 3.78(4H, t, J=4.88Hz), 5.62 (1H, s), 6.64 (1H, s), 6.73 (2H, m), 7.11 (1H,s), 8.79 (1H, d, J=7.08Hz)

ES-MS; m/z: 370 (M⁺+1)

(H)8-[2-(4-Ethyl-2-thienyl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carbaldehyde

Reactions were performed in the same manner as in Example 1, (I) byusing8-[2-(4-ethyl-2-thienyl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one(122 mg, 0.33 mmol) as a starting material. As a result, 52 mg (40% forthe three steps) of the title compound in orange color was obtained.

¹H-NMR (CDCl₃) δ: 1.20 (3H, t, J=7.56Hz), 2.56 (2H, q, J=7.56Hz), 3.02(2H, t, J=8.04Hz), 3.17 (2H, t, J=8.04Hz), 3.73 (4H, d, J=4.88Hz), 3.82(4H, d, J=4.88Hz), 6.64 (1H, s), 6.74 (1H, s), 6.76 (1H, dd, J=1.95,7.07Hz), 7.03 (1H, s), 8.74 (1H, d, J=7.07Hz), 10.11 (1H, s)

EI/MS; m/z: 398 (M⁺+1)

(I) tert-Butyl(E)-3-{8-[2-(4-ethyl-2-thienyl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoate

Reactions were performed in the same manner as in Example 1, (J) byusing8-[2-(4-ethyl-2-thienyl)ethyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde(52 mg,0.13 mmol) as a starting material. As a result, 65 mg of thetitle compound was obtained as an orange oily substance in a mixturewith triphenylphosphine oxide.

¹H-NMR (CDCl₃) δ: 1.20 (3H, t, J=7.59Hz), 1.51 (9H, s), 2.56 (2H, q,J=7.59Hz), 3.04 (2H, t, J=8.08Hz), 3.17 (2H, t, J=8.08Hz), 3.60 (4H, t,J=4.41Hz), 3.83 (4H, t, J=4.41Hz), 6.63 (1H, s), 6.73 (1H, s), 6.82 (1H,dd, J=1.96, 7.35Hz), 7.05 (1H, d, J=15.68Hz), 7.17 (1H, s), 7.69 (1H, d,J=15.68Hz), 8.87 (1H, d, J=7.35Hz)

EI/MS; m/z: 496 (M⁺+1)

(J)(E)-3-{8-[2-(4-Ethyl-2-thienyl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-2-propenoicacid

Reactions were performed in the same manner as in Example 1, (L) byusing tert-butyl(E)-3-{8-[2-(4-ethyl-2-thienyl)ethyl]-2-morpholino-.4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-2-propenoate(65 mg, 0.13 mmol) as a starting material. As a result, 32 mg (56%) ofyellow crystals were obtained.

¹H-NMR (CDCl₃) δ: 1.20 (3H, t, J=7.57Hz), 2.56 (2H, q, J=7.57Hz), 3.05(2H, t, J=7.81Hz), 3.18 (2H, t, J=7.81Hz), 3.63 (4H, t, J=4.40Hz), 3.84(4H, t, J=4.40Hz), 6.63 (1H, s), 6.74 (1H, s), 6.84 (1H, dd, J=1.95,7.32Hz), 7.11 (1H, d, J=15.38Hz), 7.18 (1H, s), 7.69 (1H, d, J=15.38Hz),8.88 (1H, d, J=7.32Hz)

EI/MS; m/z: 440 (M⁺+1)

Example 7(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A) 2-(Acetylamino)isonicotinic acid

N¹-(4-Methyl-2-pyridyl)acetamide (25 g, 0.168 mol) suspended in water(250 ml) was added with potassium permanganate (76.1 g, 0.50 mol) at100° C. over 1 hour and then stirred for 40 minutes. The reactionsolution was returned to room temperature, and black crystals wereremoved by filtration. The resulting filtrate was added with 12 Nhydrochloric acid until pH of the solution became 3 to 4. After thereaction solution was stirred for about 15 minutes, the deposited whitecrystals were collected by filtration, washed with water, and dried byusing a vacuum pump to obtain 7.65 g (25%) of the title compound withoutpurification.

¹H-NMR (CD₃OD) δ: 2.20 (3H, s), 7.59 (1H, dd, J=1.47, 5.13Hz), 8.42 (1H,dd, J=0.73, 5.13Hz), 8.63 (1H, s)

EI/MS; m/z: 179 (M⁺)

(B) N⁴-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-(acetylamino)isonicotinamide

2-(Acetylamino)isonicotinic acid (500 mg, 2.8 mmol) was added dropwisewith thionyl chloride (15 ml, 68.5 mmol) at room temperature, warmed to80° C. and stirred for 30 minutes. The reaction solution was returned toroom temperature, concentrated under reduced pressure, and the residuewas subjected to azeotropy with toluene to evaporate excessive thionylchloride. The resulting yellow crystals was added to a mixed solution ofpyridine (0.25 ml), dichloromethane (5 ml) and4-(tert-butyl)-1,3-thiazol-2-amine (525 mg, 3.35 mmol) under icecooling. The mixture was stirred at 0° C. for 30 minutes, then warmed toroom temperature, and further stirred for 2 hours and 30 minutes. Thereaction solution was added with water and extracted with chloroform.The organic layer was dried over magnesium sulfate and concentratedunder reduced pressure. The resulting residue was purified by silica gelcolumn chromatography (chloroform→chloroform:methanol=80:1→50:1) toobtain 545.8 mg (61%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.31 (9H, s), 2.26 (3H, s), 6.61 (1H, s), 7.63 (1H,dd, J=0.49, 5.14Hz), 8.44 (1H, d, J=5.14Hz), 8.51 (1H, brd), 8.72 (1H,s)

EI/MS; m/z: 319 (M⁺+1)

(C) N⁴-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2- aminoisonicotinamide

N⁴-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-(acetylamino)isonicotinamide (546mg) dissolved in ethanol (12 ml) was added dropwise with concentratedhydrochloric acid (1.2 ml) at room temperature and stirred at 80-90° C.for 1 hour. The reaction solution was returned to room temperature,concentrated under reduced pressure, neutralized with 1 N sodiumhydroxide and extracted with chloroform. The organic layer was driedover magnesium sulfate and concentrated under reduced pressure, and theresulting residue was purified by silica gel column chromatography(chloroform→chloroform:methanol=60:1 b 20:1) to obtain 247.2 mg (52%) ofthe title compound as white crystals.

¹H-NMR (CD₃OD) δ: 1.33 (9H, s), 6.63 (1H, d, J=0.73Hz), 7.05 (1H, t,J=0.73Hz), 7.09 (1H, ddd, J=0.73, 1.46, 5.36Hz), 8.13 (1H, dd, J=0.73,5.36Hz)

EI/MS; m/z: 275 (M⁺−1)

(D)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]-pyrimidine-8-carboxamide

N⁴-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-aminoisonicotinamide (200 mg,0.724 mmol) was added with xylene (20 ml) and trichlorophenyl malonate(370 mg, 0.796 mmol) and refluxed by heating at 130° C. The reactionsolution was stirred for 1 hour, then returned to room temperature andconcentrated under reduced pressure. The resulting orange crystals wastaken by filtration, washed with chloroform, dried under reducedpressure to obtain 209 mg (84%) of the title compound as orange crystalswithout purification.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 3.45 (2H, m), 6.88 (1H, s), 7.77 (1H,d, J=7.08Hz), 8.02 (1H, s), 8.99 (1H, d, J=7.08Hz)

EI/MS; m/z: 345 (M⁺+1)

(E)N⁸-[4-(tert-Butyl)-1,3thiazol-2-yl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide

Reactions were performed in the same manner as in Example 2, (A) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxamide(209 mg, 0.607 mmol). The resulting crude product was purified by silicagel column chromatography (chloroform:methanol=50:1→30:1) to obtain 147mg of the title compound in a mixture with byproducts.

¹H-NMR (CD₃OD) δ: 1.36 (9H, s), 3.46 (4H, t, J=4.64Hz), 3.58 (4H, t,J=4.64Hz), 5.71 (1H, s), 6.64 (1H, s), 7.51 (1H, d, J=7.57Hz), 7.99 (1H,s), 8.89 (1H, d, J=7.57Hz)

EI/MS; m/z: 414 (M⁺+1)

(F)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-3-formyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxamide

Dimethylformamide (1 ml) was added dropwise with phosphorus oxychloride(66 μl, 0.712 mmol) under ice cooling, then warmed to room temperature,and stirred for 15 minutes. The reaction solution was cooled to 0° C.with ice, added withN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(147 mg, 0.356 mmol) dissolved in dimethylformamide (4 ml) under icecooling, and stirred at 0° C. for 2 hours and 30 minutes. The reactionsolution was neutralized with saturated aqueous sodium hydrogencarbonateand concentrated under reduced pressure. The resulting residue was addedwith chloroform for extraction, and the organic layer was dried overmagnesium sulfate and concentrated under reduced pressure. The resultingresidue was purified by thin layer silica gel chromatography(chloroform:methanol=40:1) to obtain 98.2 mg of the title compound asyellow crystals in a mixture with dimethylformamide.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 3.78 (4H, d, J=4.88Hz), 3.82 (4H, d,J=4.88Hz), 6.59 (1H, s), 7.44 (1H, dd, J=1.71, 7.32Hz), 7.86 (1H, s),8.87 (1H, d, J=7.32Hz), 10.16 (1H, s)

EI/MS; m/z: 442 (M⁺+1)

(G) Methyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-3-formyl-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidine-8-carboxamide(98.2 mg, 0.223 mmol) was added with tetrahydrofuran (6 ml), lithiumchloride (30 mg, 0.669 mmol) andbis(2,2,2-trifluoroethyl)(methoxycarbonylmethyl)-phosphonate (142 μl,0.669 mmol), then added dropwise with 1,8-diazabicyclo[5.4.0]undec-7-ene(92 μl, 0.669 mmol) at room temperature, stirred at room temperature for1 hour, and further added withbis(2,2,2-trifluoroethyl)(methoxycarbonylmethyl)-phosphonate (75 μl) and1,8-diazabicyclo[5.4.0]undec-7-ene (45 μl). The reaction solution wasfurther stirred at room temperature for 30 minutes and concentratedunder reduced pressure. The resulting residue was purified by thin layersilica gel chromatography (chloroform:methanol=40:1) to obtain 145.8 mgof the title compound in a mixture with1,8-diazabicyclo[5.4.0]undec-7-ene.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 3.70 (4H, m), 3.78 (4H, m), 3.86 (3H,s), 6.62 (1H, s), 7.12 (1H, d, J=15.63Hz), 7.41 (1H, s), 7.58 (1H, d,J=7.57Hz), 7.59 (1H, d, J=15.63Hz), 8.98 (1H, d, J=7.57Hz)

EI/MS; m/z: 498 (M⁺+1)

(H)(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

Methyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(145.8 mg, 0.293 mmol) was added with methanol (2 ml), tetrahydrofuran(5 ml) and water (1 ml), and then added dropwise with 1 N sodiumhydroxide (1 ml) at room temperature. After the reaction solution wasstirred at room temperature for 1 hour, the solution was further addedwith 1 N sodium hydroxide (2. ml) and further stirred at roomtemperature for 15 hours. The reaction solution was added with 1 Nhydrochloric acid until pH of the solution became 4 and extracted withchloroform. The organic layer was dried over magnesium sulfate andconcentrated under reduced pressure. The resulting residue was purifiedby thin layer silica gel chromatography (chloroform:methanol=20:1) toobtain 15.4 mg (5% for the four steps) of the title compound as yellowcrystals.

¹H-NMR (DMSO-d₆) δ: 1.27 (9H, s), 3.70 (4H, m), 3.78 (4H, m), 6.54 (1H,brd), 6.93 (1H, d, J=15.38Hz), 7.48 (1H, d, J=15.38Hz), 7.78 (1H, d,J=7.08Hz), 8.05 (1H, s), 8.88 (1H, d, J=7.08Hz)

ES-MS: 484 (M⁺+1), 482 (M⁺−1)

Example 8(E)-3-[8-{[(4-Cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A) 4-Cyclobutyl-1,3-thiazol-2-amine

Thiourea (2.3 g, 30.0 mmol) was dissolved in ethanol (100 ml), addedwith 2-bromo-1-cyclobutyl-1-ethanone synthesized in the same manner asin Example 1, (E), and then heated to 100° C. The reaction solution wasstirred for 1 hour, then returned to room temperature, neutralized withsaturated sodium hydrogencarbonate and extracted with chloroform. Thecollected organic layer was washed with saturated brine, dried overmagnesium sulfate and then concentrated under reduced pressure. Thetitle compound was obtained as the residue without purification as abrown oily substance (5.4 g, 100%).

¹H-NMR (CDCl₃) δ: 1.86 (1H, m), 1.98 (1H, qu, J=9.31Hz), 2.12-2.37 (4H,m), 3.38 (1H, qu, J=8.08Hz), 5.36 (2H, brd), 6.07 (1H, s)

(B) N⁴-(4- Cyclobutyl-1,3-thiazol-2-yl)-2-(acetylamino)isonicotinamide

Reactions were performed in the same manner as in Example 7, (B) byusing 2-(acetylamino)isonicotinic acid (3.3 g, 18.42 mmol) as thestarting material and 4-cyclobutyl-1,3-thiazol-2-amine (3 g, 18.42 mmol)to obtain 3.76 g (65%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.91 (1H, m), 2.03 (1H, qu, J=9.03Hz), 2.15-2.42 (4H,m), 2.23 (3H, s), 3.55 (1H, qu, J=8.55Hz), 6.63 (1H, s), 7.56 (1H, d,J=5.13Hz), 8.37 (1H, s), 8.66 (1H, s)

EI/MS; m/z: 317 (M⁺+1)

(C) N⁴-(4-Cyclobutyl-1,3-thiazol-2-yl)-2-aminoisonicotinamide

Reactions were performed in the same manner as in Example 7, (C) byusing N⁴-(4-cyclobutyl-1,3-thiazol-2-yl)-2-(acetylamino)isonicotinamide(3.76 g, 11.9 mmol) to obtain 1.73 g (53%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.94-2.37 (6H, m), 3.58 (1H, qu, J=8.57Hz), 6.67 (1H,s), 7.07 (1H, s), 7.09 (1H, d, J=5.14Hz), 8.10 (1H, d, J=5.14Hz)

EI/MS; m/z: 273 (M⁺−1)

(D) N⁸-(4-Cyclobutyl-1,3-thiazol-2-yl)-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]-pyrimidine-8-carboxamide

Reactions were performed in the same manner as in Example 7, (D) byusing N⁴-(4-cyclobutyl-1,3-thiazol-2-yl)-2-aminoisonicotinamide (1.73 g,6.3 mmol) to obtain 1.85 g (86%) of the title compound as brown solid.

¹H-NMR (DMSO-d₆) δ: 1.85-1.98 (2H, m), 2.16-2.27 (4H, m), 3.47 (2H, m),3.57 (1H, m), 6.91 (1H, s), 7.74 (1H, d, J=7.35Hz), 8.27 (1H, s), 8.97(1H, d, J=7.35Hz)

EI/MS; m/z: 343 (M⁺+1)

(E)N⁸-(4-Cyclobutyl-1,3-thiazol-2-yl)-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido-[1,2-a]pyrimidine-8-carboxamide

N⁸-(4-Cyclobutyl-1,3-thiazol-2-yl)-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]-pyrimidine-8-carboxamide (100 mg, 0.29 mmol) was addedwith acetonitrile (2 ml) and dimethylformamide (1 ml), cooled to −10° C.with ice, then added with diphenyl chlorophosphate (0.2 ml, 0.96 mmol),and further added dropwise with diisopropylethylamine (0.3 ml, 1.74mmol). The reaction solution was stirred at −10° C. for 30 minutes, thenadded with 3-hydroxypiperidine (90 mg, 0.89 mmol), warmed to roomtemperature, and then further warmed to 80° C. As the reaction was notcompleted, the reaction solution was further added with3-hydroxypiperidine (60 mg), stirred for 1 hour, and further added with3-hydroxypiperidine (70 mg). After the disappear of the startingmaterial was observed, the reaction was stopped with saturated aqueoussodium hydrogencarbonate, and the reaction solution was extracted withchloroform. The organic layer was dried over magnesium sulfate andconcentrated under reduced pressure. The resulting residue was purifiedby thin layer silica gel chromatography (chloroform:methanol=15:1) toobtain 85.2 mg (69%) of the title compound as yellow crystals.

¹H-NMR (CDCl₃) δ: 1.60-2.06 (6H, m), 2.21-2.35 (4H, m), 3.57 (4H, m),3.95 (2H, m), 5.75 (1H, s), 6.62 (1H, s), 7.28 (1H, dd, J=1.71, 7.56Hz),7.74 (1H, s), 8.87 (1H, d, J=7.56Hz)

EI/MS; m/z: 426 (M⁺+1)

(F)N⁸-(4-Cyclobutyl-1,3-thiazol-2-yl)-3-formyl-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide

Reactions were performed in the same manner as in Example 7, (F) byusingN⁸-(4-cyclobutyl-1,3-thiazol-2-yl)-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxamide(85.2 mg, 0.20 mmol) to obtain 27.5 mg (30%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.84-2.38 (10H, m), 3.43-3.63 (4H, m), 3.90 (1H, m),4.08 (1H, m), 6.61 (1H, s), 7.39 (1H, d, J=7.07Hz), 7.82 (1H, s), 8.87(1H, d.J=7.07Hz), 10.09 (1H, s)

EI/MS; m/z: 454 (M⁺+1)

(G) Methyl (E)-3-[8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

Reactions were performed in the same manner as in Example 7, (G) byusingN⁸-(4-cyclobutyl-1,3-thiazol-2-yl)-3-formyl-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido-[1,2-a]pyrimidine-8-carboxamide(27.5 mg, 0.061 mmol) to obtain 16.3 mg (53%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.91 (5H, m), 2.06 (1H, m), 2.20 (2H, m), 2.38 (2H,m), 3.61 (4H, m), 3.68 (3H,s), 3.88 (1H, m), 4.04 (1H, m), 6.60 (1H, s),7.14 (1H, d, J=15.63Hz), 7.47 (1H.dd, J=1.95, 7.57Hz), 7.57 (1H, d,J=15.63Hz), 7.94 (1H, s), 8.99 (1H, d, J=7.57Hz)

EI/MS; m/z: 510 (M⁺+1)

(H)(E)-3-[8-{[(4-Cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

Reactions were performed in the same manner as in Example 7, (H) byusing methyl(E)-3-[8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(16.3 mg, 0.032 mmol) to obtain 11 mg (69%) of the title compound asorange crystals.

¹H-NMR (CD₃OD) δ: 1.59-2.08 (6H, m), 2.27-2.37 (4H, m), 3.62 (3H, s),3.87 (2H, m), 4.08 (1H, d, J=12.21Hz), 6.69 (1H, s), 7.09 (1H,d,J=15.87Hz), 7.48 (1H, d, J=15.87Hz), 7.60 (1H, dd, J=1.95, 7.32Hz), 8.07(1H, s), 8.94 (1H, d, J=7.32Hz)

EI/MS; m/z: 496 (M⁺+1)

Example 9(E)-3-[8-{[(4-Cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(4-methylpiperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoic acid

(A)N⁸-(4-Cyclobutyl-1,3-thiazol-2-yl)-2-(4-methylpiperazino)-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxamide

Reactions were performed in the same manner as in Example 8, (E) byusingN⁸-(4-cyclobutyl-1,3-thiazol-2-yl)-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxamide(60 mg, 0.174 mmol) and N-methylpiperazine (60 μl, 0.52 mmol) as regentsto obtain 51.2 mg (69%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.94 (1H, m), 2.04 (1H, m), 2.23 (2H, m), 2.34 (2H,m), 2.35 (3H, s), 2.50 (4H, t, J=5.13Hz), 3.57 (1H, qu, J=8.30Hz), 3.72(4H, brd), 5.68 (1H, s), 6.63 (1H, s), 7.30 (1H.dd, J=1.95, 7.32Hz),7.80 (1H, s), 8.95 (1H, d, J=7.32Hz)

EI/MS; m/z: 425 (M⁺+1)

(B) Methyl(E)-3-[8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(4-methylpiperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

Reactions were performed in the same manner as in Example 7, (F) byusingN⁸-(4-cyclobutyl-1,3-thiazol-2-yl)-2-(4-methylpiperazino)-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxamide(51.2 mg, 0.1206 mmol), and the product was used without purification toperform reactions in the same manner as in Example 7,(G) to obtain 24.7mg (40% for the two steps) of the title compound.

¹H-NMR (CDCl₃) δ: 1.94 (1H, m), 2.05 (1H, m), 2.23 (2H, m), 2.34 (3H,s), 2.35 (2H, m), 2.54 (4H, m), 3.58 (1H, qu, J=8.54Hz), 3.68 (4H, m),3.81 (3H, s), 6.62 (1H, s), 7.16 (1H, d, J=15.63Hz), 7.43 (1H, dd,J=1.95, 7.57Hz), 7.57 (1H, d, J=15.63Hz), 7.88 (1H, s), 8.99 (1H, d,J=7.57Hz)

EI/MS; m/z: 509 (M⁺+1)

(C)(E)-3-[8-{[(4-Cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(4-methylpiperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

Reactions were performed in the same manner as in Example 7, (H) byusing methyl(E)-3-3-[8-[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(4-methylpiperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(24.7 mg, 0.049 mmol) to obtain 14.1 mg (59%) of the title compound inyellow color.

¹H-NMR (CD₃OD) δ: 1.96-2.38 (6H, m), 2.37 (3H, s), 2.61 (4H, m), 3.59(1H, m), 3.67 (4H, m), 6.66 (1H, s), 7.08 (1H, d, J=15.63Hz), 7.48 (1H,d, J=15.63Hz), 7.61 (1H.dd, J=1.71, 7.57Hz), 8.09 (1H, s), 8.96 (1H, d,J=7.57Hz)

EI/MS; m/z: 495 (M⁺+1)

Example 10N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-4-oxo-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidine-8-carboxamide

(A) Ethyl 2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]acetate

Ethyl 1H-tetrazole-5-acetate (30 g, 0.192 mol) was dissolved indimethylformamide (100 ml), cooled with ice, and added with potassiumcarbonate (32 g, 0.231 mol). The mixture was added dropwise with4-methoxybenzochloride (31 ml, 0.231 mol) and stirred at roomtemperature for 21 hours. The reaction solution was concentrated underreduced pressure and subjected to azeotropy with toluene. The resultingresidue was diluted with ethyl acetate and washed with water, and thecompound dissolved in the aqueous layer was extracted with ethylacetate. The organic layer collected was dried over magnesium sulfate.The organic layer was concentrated under reduced pressure, and then theresidue was purified by silica gel column chromatography (hexane:ethylacetate=2:1) to obtain 24 g (46%) of the title compound as a colorlesstransparent substance.

¹H-NMR (CDCl₃) δ: 1.25 (3H, t, J=7.07Hz), 3.79 (3H, s), 3.93 (2H, s),4.18 (2H, qu, J=7.07Hz), 5.68 (2H, s), 6.88 (2H, d, J=8.78Hz), 7.31 (2H,d, J=8.78Hz)

EI/MS; m/z: 275 (M⁺−1)

(B) Ethyl3-(dimethylamino)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-2-propenoate

Dimethylformamide dimethylacetal (13 ml, 97.3 mmol) was dissolved inethyl 2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]acetate (24 g,88.4 mmol) and heated to 100° C. for 3 hours with stirring. The reactionsolution was purified by silica gel column chromatography (hexane:ethylacetate=3:1→1:1→1:3) to obtain 14 g (48%) of yellow crystals.

¹H-NMR (CDCl₃) δ: 1.26 (3H, t, J=7.07Hz), 2.04 (6H, s), 3.79 (3H, s),4.11 (2H, qu, J=7.0.7Hz), 5.70 (2H, s), 6.86 (2H, d, J=9.02Hz), 7.30(2H, d, J=9.02Hz), 7.73 (1H, s)

EI/MS; m/z: 332 (M⁺+1)

(C) Ethyl 2-aminoisonicotinate

2-(Acetylamino)isonicotinic acid (13 g, 73.60 mmol) synthesized by themethod of Example 7, (A) was added with ethanol (50 ml) and toluene (150ml) and heated to 100-110° C. The mixture was added dropwise withconcentrated sulfuric acid (7 ml) and heated for 11hour with stirring.The reaction solution was returned to room temperature and poured intosaturated aqueous sodium hydrogencarbonate cooled with ice. The mixturewas extracted with chloroform and the collected organic layer was driedover magnesium sulfate and concentrated under reduced pressure to obtain7.6 g (23% for the two steps) of the title compound as pale yellowcrystals without purification.

¹H-NMR (CDCl₃) δ: 1.39 (3H, t, J=7.07Hz), 4.37 (2H, qu, J=7.07Hz), 4.63(2H, brd), 7.07 (1H, s), 7.17 (1H, dd, J=0.98, 5.12Hz), 8.18 (1H, d,J=5.12Hz)

EI/MS; m/z: 165 (M⁺−1)

(D) Ethyl3-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxylate

Ethyl 2-aminoisonicotinate (6.5 g, 39.23 mmol) was added with aceticacid (500 ml) and ethyl3-(dimethylamino)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-2-propenoate(13 g, 39.23 mmol) and refluxed by heating at 130° C. for 5 hours. Thereaction solution was returned to room temperature and poured intowater, and this was extracted with chloroform. The resulting organiclayer was washed with saturated brine, and the collected organic layerwas dried over magnesium sulfate. The organic layer was concentratedunder reduced pressure and subjected to azeotropy with toluene, and theresulting residue was purified by silica gel column chromatography(chloroform→chloroform:methanol=80:1→50:1→30:1) to obtain 8.6 g of thetitle compound.

¹H-NMR (CDCl₃) δ: 1.45 (3H, t, J=7.08Hz), 3.79 (3H, s), 4.49 (2H,q,J=7.08Hz), 5.82 (2H, s)6.89 (2H, d, J=8.54Hz), 7.30 (2H, d, J=8.54Hz),7.74 (1H, d, J=6.59Hz), 8.39 (1H, s), 9.28 (1H, s), 9.29 (1H, d,J=6.59Hz)

EI/MS; m/z: 407 (M⁺+1)

(E)3-[2-(4-Methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxylicacid

Ethyl3-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxylate(8.6 g, 21.1 mmol) was dissolved in tetrahydrofuran (170 ml) and addeddropwise with 0.5 N aqueous sodium hydroxide (65 ml) at roomtemperature. The reaction solution was stirred for 1 hour and 30minutes, then added with 1 N hydrochloric acid (55 ml) and water (250ml) under ice cooling and warmed to room temperature with stirring. Thedeposited yellow crystals were taken by filtration, washed with water,dissolved in ethanol and concentrated under reduced pressure. Theresulting crystals were dried under reduced pressure to obtain 4 g (27%for the two steps) of the title compound without purification.

¹H-NMR (CDCl₃) δ: 3.81 (3H, s), 5.83 (2H, s), 6.92 (2H, d, J=8.55Hz),7.43 (2H, d, J=8.55Hz), 7.83 (1H, dd, J=1.22, 7.32Hz), 8.40 (1H, s),9.23 (1H, s), 9.31 (1H, d, J=7.32Hz)

EI/MS; m/z: 379 (M⁺+1)

(F)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-4-oxo-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido-[1,2-a]pyrimidine-8-carboxamide

3-[2-(4-Methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxylicacid (40 mg, 0.114 mmol) was added with dimethylformamide (2 ml),4-(tert-butyl)-1,3-thiazol-2-amine (20 mg, 0.125 mmol),1-hydroxybenzotriazole (20 mg, 0.136 mmol) and 4-dimethylaminopyridine(21 mg, 0.170 mmol) at room temperature, then added with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (35 mg,0.170 mmol) at room temperature, and stirred at room temperature for 24hours. The reaction solution was added dropwise with 1 N hydrochloricacid (3 ml) at room temperature, and the deposited yellow crystals weretaken by filtration and washed with water. The crystals were dissolvedin chloroform and ethanol, concentrated under reduced pressure, anddried to obtain the title compound as yellow crystals withoutpurification. The product was dissolved in anisole (0.2 ml) andtrifluoroacetic acid (5 ml), stirred at room temperature for 5 hours,and added with water. The deposited yellow crystals were collected byfiltration and washed with water, and the resulting crystals weredissolved in chloroform and ethanol and concentrated under reducedpressure. The crude crystals was added with ethanol, taken by filtrationand washed with diethyl ether to obtain 28 mg (67% for the two steps) ofthe title compound as yellow crystals.

¹H-NMR (CD₃OD) δ: 1.38 (9H, s), 6.66 (1H, s), 8.10 (1H, d, J=8.30Hz),8.57 (1H, s), 9.38 (1H, d, J=8.30Hz), 9.39 (1H, s)

EI/MS; m/z: 397 (M⁺+1)

Example 11N⁸-[4-(4-Pyridyl)-1,3-thiazol-2-yl]-4-oxo-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidine-8-carboxamide

(A) 2-Bromo-1-(4-pyridyl)-1-ethanone

4-Acetylpyridine (3.62 g, 29.9 mmol) was added with acetic acid (30 ml)and 47% hydrobromic acid (5.3 ml) at room temperature, subsequentlyadded dropwise with bromine (1.6 ml in total) four times at an intervalof 5 minutes, stirred at room temperature for 2 hours and 30 minutes.The mixture was further added with bromine (1.6 ml), and stirred at roomtemperature for 20 hours. The reaction solution was filtered, and theresulting crystals were washed with diethyl ether and dried to obtain12.43 g of the title compound as a salt of hydrogen bromide in the formof orange crystals without purification.

¹H-NMR (CD₃OD) δ: 3.73 (2H, dd, J=1.21,28.76Hz), 8.26 (2H, d, J=6.83Hz),8.89 (2H, d, J=6.83Hz)

EI/MS; m/z: 279 (M⁺−1)

(B) 4-(4-Pyridyl)-1,3-thiazol-2-amine

Reactions were performed in the same manner as in Example 8, (A) byusing 2-bromo-1-(4-pyridyl)-1-ethanone (1 g, 3.56 mmol) to obtain 410 mg(65%) of the title compound.

¹H-NMR (CD₃OD) δ: 7.26 (1H, s), 7.80 (2H, d, J=6.35Hz), 8.50 (2H, d,J=6.35Hz)

EI/MS; m/z: 176 (M⁺−1)

(C)N⁸-[4-(4-Pyridyl)-1,3-thiazol-2-yl]-4-oxo-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido-[1,2-a]pyrimidine-8-carboxamide

Reactions were performed in the same manner as in Example 10, (F) byusing3-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (120 mg, 0.32 mmol) and 4-(4-pyridyl)-1,3-thiazol-2-amine (62 mg,0.35 mmol) to obtain 35.7 mg (27% for the two steps) of the titlecompound as orange crystals.

¹H-NMR (DMSO-d₆) δ: 8.03 (3H, m), 8.29 (1H, s), 8.66 (1H, d, J=0.977Hz),8.73 (2H, d, J=5.62Hz), 9.29.(1H, s), 9.32 (1H, d, J=6.54Hz)

ES-MS; m/z: 418 (M⁺+1)

IR (cm⁻¹): 1668, 1633, 1567, 1492, 1288

Example 12N⁸-(1,3-Benzothiazol-2-yl)-4-oxo-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido-[1,2-a]pyrimidine-8-carboxamide

Reactions were performed in the same manner as in Example 10, (F) byusing3-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (60 mg, 0.16 mmol) and 2-aminobenzothiazole (30 mg, 0.17 mmol) toobtain 2 mg (3% for the two steps) of an orange compound.

¹H-NMR (DMSO-d₆) δ: 7.39 (1H, t, J=7.57Hz), 7.52 (1H, t, J=7.57Hz), 7.78(1H, d, J=7.57Hz), 8.05 (2H, d, J=7.57Hz), 8.61 (1H, s), 9.28 (1H, s),9.31 (1H, d, J=7.57Hz)

ES-MS; m/z: 391 (M⁺+1)

Example 13N⁸-(4-Cyclobutyl-1,3-thiazol-2-yl)-2-(4-methylpiperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide

Reactions were performed in the same manner as in Example 9, (A) byusingN⁸-(4-cyclobutyl-1,3-thiazol-2-yl)-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxamide(40 mg, 0.12 mmol), and the resulting compound was added with 4 Nhydrochloric acid solution in dioxane (2 ml) and stirred to obtain 12 mg(24%) of the title compound as hydrochloride.

NMR for free form and Mass:

¹H-NMR (CDCl₃) δ: 1.94 (1H, m), 2.04 (1H, m), 2.23 (2H, m), 2.34 (2H,m), 2.35 (3H,s), 2.50 (4H, t, J=5.13Hz), 3.57 (1H, qu, J=8.30Hz), 3.72(4H, brd), 5.68 (1H, s), 6.63 (1H, s), 7.30 (1H.dd, J=1.95, 7.32Hz),7.80 (1H, s), 8.95 (1H, d, J=7.32Hz)

EI/MS; m/z: 425 (M⁺+1)

Example 14N⁸-(4-Isopropyl-1,3-thiazol-2-yl)-4-oxo-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidine-8-carboxamide

(A) N⁸-(4-Isopropyl-1,3-thiazol-2-yl)-3-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide

3-[2-(4-Methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxylicacid (250 mg, 0.66 mmol) was dissolved in dimethylformamide (6 ml),added with N,N-carbonylbis-1H-imidazole (abbreviated as “CDI”hereinafter, 160 mg, 0.991 mmol) and heated to 90° C. with stirring.After 1 hour and 30 minutes, the reaction solution was further addedwith CDI (170 mg), further stirred for 1 hour, and then further addedwith CDI (170 mg). The reaction solution was stirred further 2 hours,then returned to room temperature, added with4-isopropyl-1,3-thiazol-2-amine hydrobromide (1.32 mmol) and stirred atroom temperature for 2 hours and 30 minutes. The reaction solution wasadded with 2 N hydrochloric acid (7 ml), and the deposited crystals werecollected by filtration and washed with water. The resulting yellowcrystals were dissolved in chloroform and washed with saturated brine,and the collected organic layer was dried over magnesium sulfate andconcentrated under reduced pressure to obtain the title compound asyellow crystals without purification.

¹H-NMR (CD₃OD) δ: 1.34 (6H, d, J=6.83Hz), 3.00 (1H, qu, J=6.83Hz), 3.80(3H, s), 5.85 (2H, s), 6.63 (1H, s), 6.92 (2H, d, J--8.78Hz), 7.44 (2H,d, J=8.78Hz), 8.04 (1H, dd, J=1.46, 7.56Hz), 8.50 (1H, s), 9.20 (1H, s),9.34 (1H, d, J=7.56Hz)

EI/MS; m/z: 503 (M⁺+1)

(B)N⁸-(4-Isopropyl-1,3-thiazol-2-yl)-4-oxo-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido-[1,2-a]pyrimidine-8-carboxamide

N⁸-(4-Isopropyl-1,3-thiazol-2-yl)-3-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(330 mg, 0.66 mmol) was added with trifluoroacetic acid (10 ml) at roomtemperature and stirred for 3 days. The reaction solution was pouredinto ice water, and the deposited yellow crystals were collected byfiltration, washed with water, then dissolved in chloroform and ethanol,and concentrated under reduced pressure. The resulting yellow crystalswere suspended in a small amount of ethanol, and the crystals were takenby filtration and washed with diethyl ether to obtain 54 mg (21% for thetwo steps) of the title compound as yellow crystals.

¹H-NMR (DMSO-d₆) δ: 1.27 (6H, d, J=6.84Hz), 2.93 (1H, m), 6.90 (1H, s),8.02 (1H, d, J=7.08Hz), 8.55 (1H, s), 9.26 (1H, s), 9.29 (1H, d,J=7.08Hz)

ES-MS; m/z: 383 (M⁺+1)

Example 15(E)-3-(2-{3-[(Aminocarbonyl)oxy]piperidino}-8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A)1-(8-{[(4-Cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-3-formyl-4-oxo-4H-pyrido-[1,2-a]pyrimidin-2-yl)-3-piperidylformate

Reactions were performed in the same manner as in Example 7, (F) byusingN⁸-(4-cyclobutyl-1,3-thiazol-2-yl)-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxamide(85 mg, 0.20 mmol) to obtain 21 mg (22%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.92-2.41 (10H, m), 3.58-3.68 (3H, m), 3.93 (2H, m),5.10 (1H, m), 6.61 (1H, s), 7.39 (1H, dd, J=1.95, 7.31Hz), 7.79 (1H, d,J=1.95Hz), 8.01 (1H, s), 8.89 (1H, d, J=7.31Hz), 10.14 (1H, s)

EI/MS; m/z: 482 (M⁺+1)

(B) tert-Butyl(E)-3-[8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(3-formyloxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

Reactions were performed in the same manner as in Example 1, (J) byusing1-(8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-3-formyl-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)-3-piperidylformate (36 mg, 0.075 mmol), and the resulting reaction solution waspurified by thin layer silica gel chromatography(chloroform:methanol=30:1) to obtain 35 mg of the title compound asorange crystals in a mixture with triphenylphosphine oxide.

¹H-NMR (CDCl₃) δ: 1.52 (9H, s), 1.94-2.00 (6H, m), 2.23-2.34 (4H, m),3.57-3.83 (5H, m), 5.15 (1H, m), 6.62 (1H, s), 7.09 (1H, d, J=15.63Hz),7.48 (1H, dd, J=1.71, 7.32Hz), 7.49 (1H, d, J=15.63Hz), 7.91 (1H, s),8.09 (1H, s), 8.98 (1H, d, J=7.32Hz)

(C) tert-Butyl (E)3-[8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

Reactions were performed in the same manner as in Example 1, (K) byusing tert-butyl(E)-3-[8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(3-formyloxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(35 mg, 0.060 mmol), and the resulting crude crystals were purified bythin layer silica gel chromatography (chloroform:methanol=40:1) toobtain 16 mg (40% for the two steps) of the title compound as yellowcrystals.

¹H-NMR (CDCl₃) δ: 1.52 (9H, s), 1.72-2.07 (6H, m), 2.17-2.38 (4H, m),3.60 (4H, m), 3.80 (1H, m), 4.02 (1H, m), 6.61 (1H, s), 7.10 (1H, d,J=15.60Hz), 7.48 (1H, dd, J=1.71, 7.31Hz), 7.49 (1H, d, J=15.60Hz), 7.97(1H, s), 9.00 (1H, d, J=7.31Hz)

EI/MS; m/z: 552 (M⁺+1)

(D) tert-Butyl(E)-3-(2-{3-[(aminocarbonyl)oxy]piperidino}-8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

tert-Butyl(E)-3-[8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(16 mg, 0.029 mmol) was dissolved in ethyl acetate (3 ml), added withtrichloroacetyl isocyanate (10 μl) under ice cooling, stirred at 0° C.for 1 hour, then further added with trichloroacetyl isocyanate (10 μl),and further stirred for 30 minutes. Then, the reaction solution wasconcentrated under reduced pressure, and the resulting residue was addedwith methanol (2 ml), water (0.2 ml) and sodium formate (12 mg) at roomtemperature and stirred at room temperature for 7 hours. The reactionsolution was concentrated under reduced pressure, and the residue waspurified by thin layer silica gel chromatography(chloroform:methanol=30:1) to obtain 20 mg of the title compound.

¹H-NMR (CDCl₃) δ: 1.51 (9H, s), 1.90-2.36 (10H, m), 3.36-3.77 (4H, m),4.04 (1H, m), 4.92 (1H, m), 6.62 (1H, s), 7.10 (1H, d, J=15.38Hz), 7.54(1H, d, J=7.57Hz), 7.60 (1H, d, J=15.38Hz), 8.07 (1H, s), 9.00 (1H, d,J=7.57Hz)

EI/MS; m/z: 595 (M⁺+1)

(E)(E)-3-(2-{3-[(Aminocarbonyl)oxy]piperidino}-8-{[(4-cyclobutyl-1,3-thiazol-2-yl)-amino]carbonyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

tert-Butyl (E)3-(2-{3-[(aminocarbonyl)oxy]piperidino}-8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate(20 mg, 0.034 mmol) was added with a mixed solution of 4 N hydrochloricacid and dioxane (1.5 ml) at room temperature and stirred for 2 hours.The reaction solution was concentrated under reduced pressure, and theresulting residue was purified by thin layer silica gel chromatography(chloroform:methanol=10:1) to obtain 9 mg (58% for the two steps) of thetitle compound as yellow crystals.

¹H-NMR (CD₃OD) δ: 1.68 (2H, m), 1.94-2.10 (4H, m), 2.23 (2H, m), 2.38(2H, m), 3.39-3.72 (4H, m), 3.98 (1H, dd, J=5.86, 11.70Hz), 4.86 (1H,m), 6.64 (1H, s), 7.10 (1H, d, J=15.63Hz), 7.56 (1H, d, J=7.32Hz), 7.68(1H, d, J=15.63Hz), 8.07 (1H, s), 8.96 (1H, d, J=7.32Hz)

ES-MS; m/z: 539 (M⁺+1)

Example 162-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-1-cyclopropanecarboxylicacid

(A)3-[(E)-3-Hydroxy-1-propenyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one

(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoicacid (222.7 mg, 0.49 mmol) dissolved in tetrahydrofuran (8 ml) was addedwith triethylamine (342 μl) and ethyl chloroformate (141 μl) at −20° C.,then stirred at room temperature for 1 hour, and subsequently added withaqueous sodium borohydride (0.8 M, 4 ml) at room temperature. Theaqueous sodium borohydride was occasionally added until the reaction wascompleted, and the completion of the reaction was confirmed by TLC.Then, the reaction solution was added with water and extracted withchloroform and chloroform:methanol=10:1, and the organic layer collectedwas dried over magnesium sulfate and concentrated under reducedpressure. The resulting residue was purified by silica gel columnchromatography (chloroform→chloroform:methanol=100:1→60:1→30:1) torecover 81 mg (37%) of the title compound and 82 mg of the startingmaterial.

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.83Hz), 3.07 (1H, qu, J=6.83Hz), 3.19(2H, t, J=7.80Hz), 3.36 (2H, t, J=7.80Hz), 3.53 (4H, t, J=4.88Hz), 3.80(4H, t, J=4.88Hz), 4.35 (2H, d, J=5.85Hz), 6.44 (1H, d, J=15.60Hz), 6.72(1H, s), 6.83 (1H, dd, J=1.95, 7.31Hz), 7.00 (1H, dt, J=5.85, 15.60Hz)7.21 (1H, s), 8.87 (1H, d, J=7.31Hz)

EI/MS; m/s: 441 (M⁺+1)

(B)3-[2-(Hydroxymethyl)cyclopropyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one

3-[(E)-3-Hydroxy-1-propenyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one(81 mg, 0.18 mmol) was added with dichloromethane (3 ml) and a solutionof diethyl zinc in hexane (1.02 M, 270 μl, 0.275 mmol) and subsequentlyadded dropwise with iodomethane (30 μl, 0.366 mmol) at room temperature.After the reaction solution was stirred for 2 hours, the reaction wasstopped with saturated aqueous ammonium chloride, and the reactionsolution was extracted with chloroform. The organic layer was dried overmagnesium sulfate and concentrated under reduced pressure. The resultingresidue was purified by thin layer silica gel chromatography(chloroform:methanol=15:1) to obtain 61 mg (73%) of the title compound.

¹H-NMR (CDCl₃) δ: 0.90 (1H, m), 1.04 (2H, m), 1.29 (6H, d, J=6.84Hz),1.52 (1H, dt, J=5.62, 7.57Hz), 3.07 (1H, qu, J=6.84Hz), 3.17 (2H, t,J=7.32Hz), 3.35 (2H, t, J=7.32Hz), 3.56 (2H, m), 3.84 (6H, m), 4.12 (1H,d, J=9.52Hz), 4.68 (1H, brd), 6.73 (1H, s), 6.78,(1H, dd, J=1.71,7.32Hz), 7.16 (1H, s), 8.80 (1H, d, J=7.32Hz)

EI/MS; m/s: 455 (M⁺+1)

(C)2-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-1-cyclopropanecarboxylicacid

3-[2-(Hydroxymethyl)cyclopropyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one(55 mg, 0.121 mmol) dissolved in acetone (10 ml) was added dropwise withJones' reagent (130 μl, 0.363 mmol) under ice cooling with and stirredfor 2 hours. Then, the reaction solution was further added with Jones'reagent (165 μl) and stirred for 2 hours. Then, the reaction was stoppedwith saturated sodium thiosulfate, and the reaction solution wasextracted with chloroform. The organic layer was dried over magnesiumsulfate and concentrated under reduced pressure. The resulting residuewas purified by thin layer silica gel chromatography(chloroform:methanol=10:1) to obtain 2.2 mg (4%) of the title compoundas yellow crystals.

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=7.07Hz), 1.35 (1H, dt, J=4.39, 7.80Hz),1.63 (1H, dt, J=4.39, 8.53Hz), 1.85 (1H, dt, J=3.90, 8.53Hz), 2.34 (1H,dt, J=3.90, 7.80Hz), 3.07 (1H, qu, J=7.07Hz), 3.18 (2H, t, J=7.07Hz),3.35 (2H, t, J=7.07Hz), 3.58 (2H, m), 3.77 (6H, m), 6.72 (1H, s), 6.80(1H, dd, J=1.46, 7.31Hz), 7.17 (1H, s), 8.81 (1H, d, J=7.31Hz)

EI/MS; m/s: 469 (M⁺+1)

Example 17(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine -8-carboxamide

Reactions were performed in the same manner as in Example 8, (E) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxamide(300 mg, 0.871 mmol) and (R)-(+)-3-hydroxypiperidine (530 mg, 5.226mmol) to obtain 241.8 mg (65%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.57 (1H, m), 1.76 (1H, m), 1.96 (2H,m), 3.61 (3H, m), 3.90 (2H, m), 5.69 (1H, s), 6.60 (1H, s), 7.31 (1H,dd, J=1.71, 7.32Hz), 7.76 (1H, s), 8.86 (1H, d, J=7.32Hz)

EI/MS; m/s: 428 (M⁺+1)

(B)(3R)-1-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-formyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl]hexahydro-3-pyridinylformate

Reactions were performed in the same manner as in Example 7, (F) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(295 mg, 0.690 mmol) and phosphorus oxychloride (130 μl, 1.38 mmol) toobtain 277 mg (68%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.70 (1H, m), 1.87-2.03 (3H, m), 3.66(2H, m), 3.91 (2H, m), 5.09 (1H, brd), 6.58 (1H, s), 7.52 (1H, dd,J=1.71, 7.32Hz), 7.96 (1H, s), 8.01 (1H, s), 8.87 (1H, d, J=7.32Hz),10.14 (1H, s)

EI/MS; m/s: 484 (M⁺+1)

(C) tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-formyloxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

Reactions were performed in the same manner as in Example 1, (J) byusing(3R)-1-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-formyl-4-oxo-4H-pyrido-[1,2-a]pyrimidin-2-yl]hexahydro-3-pyridinylformate (277 mg, 0.5723 mmol), and the resulting residue was purified bysilica gel column chromatography(chloroform→chloroform:methanol=100:1→70:1→50:1) to obtain 559 mg (100%or more) of the title compound as a brown oily substance in a mixturewith byproducts.

¹H-NMR (CDCl₃) δ: 1.28 (9H, s), 1.53 (9H, s), 1.71-1.97 (4H, m),3.46-3.73 (4H, m), 5.09 (1H, brd), 6.56 (1H, s), 7.09 (1H, d,J=15.60Hz), 7.64 (1H, dd, J=1.95, 7.31Hz), 7.65 (1H, d, J=15.60Hz), 8.01(1H, s), 8.05 (1H, s), 8.82 (1H, d, J=7.31Hz)

EI/MS; m/s: 580 (M⁺−1)

(D) tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

Reactions were performed in the same manner as in Example 1, (K) byusing tert-butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-formyloxykexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(559 mg, 0.961 mmol) to obtain 368 mg (69%). of the title compound as anorange oily substance.

¹H-NMR (CDCl₃) δ: 1.32 (9H, s), 1.52 (9H, s), 1.77-1.90 (4H, m),3.54-3.73 (4H, m), 4.01 (1H, brd), 6.54 (1H, s), 7.04 (1H, d,J=15.63Hz), 7.64 (1H, dd, J=1.95, 7.32Hz), 7.65 (1H, d, J=15.63Hz), 8.00(1H, s), 8.84 (1H, d, J=7.32Hz)

(E)(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

Reactions were performed in the same manner as in Example 15, (E) byusing tert-butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(140 mg, 0.253 mmol) as a mixture containing triphenylphosphine oxide toobtain 42 mg (33%) of the title compound as orange crystals.

¹H-NMR (CD₃OD) δ: 1.36 (9H, s), 1.61 (2H, m), 1.92 (1H, m), 2.06 (1H,m), 3.21 (1H, dd, J=8.55, 12.94Hz), 3.36 (1H, m), 3.86 (2H, m), 4.10(1H, d, J=12.94Hz), 6.68 (1H, s), 7.05 (1H, d, J=15.63Hz), 7.60 (1H, dd,J=1.95, 7.32Hz), 7.61 (1H, d, J=15.63Hz), 8.05 (1H, s), 8.95 (1H, d,J=7.32Hz)

ES-MS; m/s: 498 (M⁺+1)

Example 18 (E)3-[2-{(3R)-3-[(Aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-([4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A) tert-Butyl(E)-3-[2-{(3R)-3-[(aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

Reactions were performed in the same manner as in Example 15, (D) byusing tert-butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(240 mg, 0.436 mmol) to obtain 254 mg (98%) of an orange oily compound.

¹H-NMR (CDCl₃) δ: 1.30 (9H, s), 1.49 (9H, s), 1.69-2.17 (4H, m), 3.34(1H, m), 3.54 (1H, m), 3.65 (1H, m), 3.87 (1H, m), 4.93 (1H, brd), 6.60(1H, s), 7.07 (1H, d, J=15.63Hz), 7.56 (1H, dd, J=1.95, 7.33Hz), 7.68(1H, d, J=15.63Hz), 8.06 (1H, s), 8.96 (1H, d, J=7.33Hz)

(B)(E)-3-[2-{(3R)-3-[(Aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

Reactions were performed in the same manner as in Example 15, (E) byusing tert-butyl(E)-3-[2-{(3R)-3-[(aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(254 mg, 0.426 mmol) containing byproducts to obtain 51 mg (22%) of thetitle compound as orange crystals.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.68 (1H, m), 1.94 (3H, m), 3.46 (1H,m), 3.66 (3H, m), 3.98 (1H, dd, J=4.88, 13.43Hz), 4.87 (1H, s), 6.63(1H, s), 7.10 (1H, d, J=15.63Hz), 7.56 (1H, dd, J=1.95, 7.33Hz), 7.68(1H, d, J=15.63Hz), 8.06 (1H, s), 8.96 (1H, d, J=7.33Hz)

EI/MS; m/s: 541 (M⁺+1)

Example 19(E)-3-[2-{(3S)-3-[(Aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3S)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide

Reactions were performed in the same manner as in Example 8, (E) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxamide(600 mg, 1.74 mmol) and (S)-(−)-3-hydroxypiperidine hydrochloride (360mg, 2.61 mmol) to obtain 463 mg (62%) of the title compound.

¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 1.59 (1H, brd), 1.73-1.99 (3H, m), 3.61(3H, m), 3.93 (2H, m), 5.71 (1H, s), 6.61 (1H, s), 7.31 (1H, d,J=7.32Hz), 7.77 (1H, s), 8.89 (1H, d, J=7.32Hz)

EI/MS; m/s: 428 (M⁺+1)

(B)(3S)1-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-formyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl]hexahydro-3-pyridinylformate

Reactions were performed in the same manner as in Example 7, (F) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-[(3S)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(463 mg, 1.08 mmol) and phosphorus oxychloride (0.3 ml, 3.25 mmol) toobtain 600 mg (100%) of the title compound as a substance containingdimethylformamide without purification.

¹H-NMR (CDCl₃) δ: 1.32 (9H, s), 1.72 (1H, m), 1.89-2.04 (3H, m), 3.68(2H, m), 3.92 (2H, d, J=3.90Hz), 5.09 (1H, brd), 6.59 (1H, s), 7.47 (1H,dd, J=1.95, 7.31Hz), 7.89 (1H, s), 8.01 (1H, s), 8.88 (1H, d, J=7.31Hz),10.14 (1H, s)

(C) tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3S)3-formyloxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

Reactions were performed in the same manner as in Example 1, (J) byusing(3S)-1-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-formyl-4-oxo-4H-pyrido-[1,2-a]pyrimidin-2-yl]hexahydro-3-pyridinylformate (524 mg, 1.083 mmol), and the resulting residue was purified bysilica gel column chromatography(chloroform→chloroform:methanol=100:1→70:1→50:1) to obtain 742 mg (100%or more) of the title compound as a brown oily substance in a mixturecontaining byproducts.

¹H-NMR (CDCl₃) δ: 1.28 (9H, s), 1.53 (9H, s), 1.68-2.03 (4H, m),3.48-3.71 (4H, m), 5.10 (1H, m), 6.57 (1H, s), 7.09 (1H, d, J=15.63Hz),7.64 (1H, dd, J=1.95, 7.32Hz), 7.65 (1H, d, J=15.63Hz), 7.99 (1H, s),8.06 (1H, s), 8.85 (1H, d, J=7.32Hz)

EI/MS; m/s: 580 (M⁺−1)

(D) tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3S)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

Reactions were performed in the same manner as in Example 1, (K) byusing tert-butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3S)-3-formyloxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(630 mg, 1.083 mmol) to obtain 439 mg (73%) of the title compound as anorange oily substance.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.52 (9H, s), 1.78-1.90 (4H, m), 3.54(2H, brd), 3.65-3.74 (2H, m), 4.02 (1H, brd), 6.53 (1H, s), 7.03 (1H, d,J=15.60Hz), 7.56 (1H, d, J=7.31Hz), 7.60 (1H, d, J=15.60Hz), 7.99 (1H,s), 8.84 (1H, d, J=7.31Hz)

EI/MS; m/s: 554 (M⁺+1)

(E) tert-Butyl(E)-3-[2-{(3S)-3-[(aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a1pyrimidin-3-yl-2-propenoate

Reactions were performed in the same manner as in Example 15, (D) byusing tert-butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3S)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(140 mg, 0.2529 mmol) to obtain 131 mg (87%) of an orange oily compound.

¹H-NMR (CDCl₃) δ: 1.30 (9H, s), 1.49 (9H, s), 1.70-2.00 (4H, m), 3.35(1H, m), 3.54 (1H, d, J=12.43Hz), 3.67 (1H, m), 3.88 (1H, m), 4.92 (1H,brd), 6.60 (1H, s), 7.05 (1H, d, J=15.60Hz), 7.55 (1H, dd, J=1.95,7.31Hz), 7.68 (1H, d, J=15.60Hz), 8.05 (1H, s), 8.97 (1H, d, J=7.31Hz)

EI/MS; m/s: 597 (M⁺+1)

(F)(E)-3-[2-{(3S)-3-[(Aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

Reactions were performed in the same manner as in Example 15, (E) byusing tert-butyl(E)-3-[2-{(3S)-3-[(aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(131 mg, 0.219 mmol) containing by products to obtain 40 mg (3.4%) ofthe title compound as orange crystals.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.68 (1H, m), 1.93 (3H, m), 3.49 (1H,m), 3.69 (2H, m), 3.96 (1H, dd, J=5.36, 12.92Hz), 4.86 (1H, brd), 6.62(1H, s), 7.11 (1H, d, J=15.60Hz), 7.55 (1H, dd, J=1.95, 7.56Hz), 7.68(1H, d, J=15.60Hz), 8.06 (1H, s), 8.96 (1H, d, J=7.56Hz)

EI/MS; m/s: 541 (M⁺+1)

Example 20(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3S)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

Reactions were performed in the same manner as in Example 15, (E) byusingtert-butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3S)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(50 mg, 0.090 mmol) containing triphenylphosphine oxide as a mixture toobtain 17 mg (38%) of the title compound as orange crystals.

¹H-NMR (CD₃OD) δ: 1.39 (9H, s), 1.65 (2H, m), 1.92 (1H, m), 2.04 (1H,m), 3.82-4.03 (5H, m), 6.62 (1H, s), 7.07 (1H, d, J=15.43Hz), 7.56 (1H,d, J=7.35Hz), 7.60 (1H, d, J=15.43Hz), 8.03 (1H, s), 8.95 (1H, d,J=7.35Hz)

ES-MS; m/s: 498 (M⁺+1)

Example 21(E)-3-{2-[(3R)-3-(Dimethylamino)tetrahydro-1H-1-pyrrolyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

tert-Butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-[(4-methylphenyl)-sulfonyl]oxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenate(31.4 mg, 0.053 mmol) was dissolved in dimethylformamide (1 ml), addedwith (3R)-(+)-3-(dimethylamino)pyrrolidine (30.1 mg, 0.26 mmol), andstirred at room temperature for 4 hours. The solvent was evaporated, andthe residue was purified by preparative TLC (chloroform:methanol=10:1,v/v) to obtain 16.8 mg of yellow oil.

The product was added with 4 N hydrochloric acid solution in dioxane (2ml), and stirred at room temperature for 4 hours. The solvent wasevaporated, and the residue was purified by preparative TLC(chloroform:methanol=10:1, v/v). The residue was lyophilized to obtain11.3 mg (44.5% for the two steps) of the title compound as yellowpowder.

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.8Hz), 1.68-1.80 (1H, m), 2.03-2.15(1H, m), 2.19 (6H, s), 2.62-2.75 (1H, m), 2.92-3.01 (1H, m), 3.14 (2H,t, J=7.7Hz), 3.49-3.58 (1H, m), 3.62-3.78 (3H, m), 6.79 (1H, d,J=15.1Hz), 7.07 (1H, s), 7.08 (1H, dd, J=7.3, 1.7Hz), 7.22 (1H, s), 7.68(1H, d, J=15.1Hz), 8.72 (1H, d, J=7.3Hz)

ESI/MS; m/z: 482 (MH⁺)

EI/MS; m/z: 481 (M⁺)

FAB/MS; m/z: 482 (MH⁺)

H-R FAB/MS: Calcd. for C₂₅H₃₁N₅/O₃S: 482.2226, Found: 482.2230

In a similar manner, the following compounds were synthesized in whichthe substituent at the 2-position was converted.

Example 22(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-[3-(oxymethyl)-piperidino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.9Hz), 1.15-1.32 (2H, m), 1.52-1.83(5H, m), 2.85 (1H, t, J=11.4Hz), 2.90-3.10 (2H, m), 3.16 (2H, t,J=7.3Hz), 3.85 (1H, brd, J=11.8Hz), 4.01 (1H, brd, J=12.0Hz), 4.56 (1H,brs), 6.85 (1H, d, J=15.4Hz), 7.07 (1H, s), 7.15 (1H, dd, J=7.3Hz), 7.28(1H, s), 7.42 (1H, d, J=15.7Hz), 8.75 (1H, d, J=7.1Hz)

ESI/MS; m/z: 483 (MH⁺)

FAB/MS; m/z: 483 (MH⁺), 505 (M⁺+Na)

Anal. Calcd. for C₂₅H₃₀N₄O₄Si.5/4H₂O: C, 50.45; H, 6.49; N, 11.09.Found: C, 50.35; H, 6.16; N, 10.68.

Example 23(E)-3-{2-[2-(Hydroxymethyl)morpholino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=7.1Hz), 2.91-3.02 (2H, m), 3.12-3.22(4H, m), 3.42-3.67 (2H, m), 3.75-3.82 (1H, m), 3.87-3.97 (2H, m),4.77-4.82 (1H, m), 6.87 (1H, d, J=15.5Hz), 7.07 (1H, s), 7.20 (1H, dd,J=7.3, 2.0Hz), 7.34 (1H, s), 7.46 (1H, d, J=15.5Hz), 8.79 (1H, d,J=7.3Hz), 11.88 (1H, brs)

ESI/MS; m/z: 485 (MH⁺)

FAB/MS; m/z: 485 (MH⁺)

H-RFAB/MS: Calcd. for C₂₄H₂₈N₄O₅S: 485.1859, Found: 485.1862

Example 24(E)-3-{2-(3-Hydroxytetrahydro-1H-1-pyrrolyl)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.9Hz), 1.78-2.00 (2H, m), 2.92-3.02(1H, m), 3.10-3.18 (3H, m), 3.55-3.64 (1H, m), 3.78-2.92 (2H, m),4.39-4.46 (1H, m), 5.00 (1H, brd, J=3.4Hz), 6.79 (1H, d, J=15.2Hz), 7.06(1H, d, J=1.7Hz), 7.07 (1H, dd, J=7.3, 0.7Hz), 7.18 (1H, d, J=1.7Hz),7.68 (1H, d, J=15.2Hz), 8.70 (1H, d, J=7.3Hz)

EI/MS; m/z: 436 (M+−H₂O)

FAB/MS; m/z: 455 (MH⁺), 477 (M⁺+Na)

H-R FAB/MS: Calcd. for C₂₃H₂₆N₄O₄S: 455.1753, Found: 455.1753

Example 25(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-[(3-pyridylmethyl)-amino]-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.19 (6H, d, J=6.8Hz), 2.90-3.10 (1H, m), 3.13 (2H,t, J=7.5Hz), 4.69 (2H, d, J=5.9Hz), 7.06 (1H, s), 7.10 (1H, dd, J=7.3,2.0Hz), 7.13 (1H, d, J=14.9Hz), 7.21 (1H, s), 7.32 (1H, dd, J=7.8,4.9Hz), 7.74 (1H, d, J=14.9Hz), 7.75 (1H, brs), 8.32-8.39 (1H, m),8.40-8.47 (1H, m), 8.58-8.62 (1H, m), 8.73 (1H, d, J=7.3Hz), 11.82 (1H,brs)

EI/MS; m/z: 431 (M⁺−CO₂)

FAB/MS; m/z: 476 (MH⁺)

H-R FAB/MS: Calcd. for C₂₅H₂₅N₅O₃S: 476.1756, Found: 476.1757

Example 26(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.19 (6H, d, J=6.8Hz), 1.30-1.48 (1H, m), 1.48-1.62(1H, m), 1.73-1.88 (1H, m), 1.88-1.98 (1H, m), 2.92-3.01 (2H, m),3.10-3.21 (3H, m), 3.37 (2H, t, J=7.6Hz), 3.54-3.65 (1H, m), 3.65-3.75(1H, m), 3.84-3.92 (1H, m), 3.85-3.63 (1H, m), 6.85 (1H, d, J=15.5Hz),7.07 (1H, s), 7.15 (1H, dd, J=7.4, 1.7Hz), 7.28 (1H, brs), 7.42 (1H, d,J=15.5Hz), 8.75 (1H, d, J=7.4Hz)

FAB/MS; m/z: 469 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₈N₄O₄S: 469.1910, Found: 469.1927

Example 27(E)-3-{2-[(3R)-3-Aminotetrahydro-1H-1-pyrrolyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=6.8Hz), 2.00-2.10 (1H, m), 2.17-2.27(1H, m), 2.93-3.03 (1H, m), 3.16 (2H, t, J=7.2Hz), 3.65-3.75 (2H, m),3.84-3.98 (3H, m), 6.86 (1H, d, J=15.2Hz), 7.09 (1H, s), 7.13 (1H, d,J=7.3Hz), 7.25 (1H, s), 7.70 (1H, d, J=15.2Hz), 8.13-7.28 (2H, br), 8.75(1H, d, J=7.3Hz)

LC/MS; m/z: 454 (MH⁺)

FAB/MS; m/z: 454 (MH⁺)

H-R FAB/MS: Calcd. for C₂₃H₂₇N₅O₃S: 454.1913, Found: 454.1920

Example 28(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-piperazino-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.8Hz), 2.92-3.02 (1H, m), 3.13-3.27(6H, m), 3.21 (2H, t, J=7.2Hz), 6.92 (1H, d, J=15.6Hz), 7.10 (1H, s),7.27 (1H, d, J=7.1Hz), 7.40 (1H, s), 7.43 (1H, d, J=15.6Hz), 8.85 (1H,d, J=7.3Hz), 9.17 (1H, br)

LC/MS; m/z: 454 (MH⁺)

EI/MS; m/z: 453 (M⁺)

FAB/MS; m/z: 454 (MH⁺), 476 (M⁺+Na)

H-R FAB/MS: Calcd. for C23H₂₇N₅O₃S: 454.1913, Found: 454.1912

Example 29(E)-3-{2-(3,5-cis-Dimethylpiperazino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)-ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=6.9Hz), 1.26 (3H, s), 1.27 (3H, s),2.92-3.02 (1H, m), 3.12-3.25 (4H, m), 3.90-3.98 (2H, m), 6.90 (1H, d,J=15.7Hz), 7.09 (1H, s), 7.26 (1H, dd, J=7.3, 1.7Hz), 7.42 (1H, d,J=15.7Hz), 7.41 (1H, s), 8.83 (1H, d, J=7.3Hz), 9.03 (1H, d, J=9.6Hz),9.50 (1H, br)

EI/MS; m/z: 481 (M⁺)

H-R EI/MS: Calcd. for C₂₅H₃₁N₅O₃S: 481.2148, Found: 481.2150

Example 30(E)-3-{2-[4-(Dimethylamino)piperidino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=6.8Hz), 1.64-1.78 (2H, m), 2.10-2.19(2H, m), 2.73 (3H, s), 2.75 (3H, s), 2.93-3.02 (1H, m), 3.03-3.14 (2H,m), 3.19 (2H, t, J=7.2Hz), 3.38 (2H, t, J=7.2Hz), 4.02-4.11 (1H, m),6.91 (1H, d, J=15.5Hz), 7.09 (1H, s), 7.22 (1H, dd, J=7.3, 1.7Hz), 7.34(1H, s), 7.43 (1H, d, J=15.5Hz), 8.81 (1H, d, J=7.3Hz), 10.19-10.27 (1H,m)

FAB/MS; m/z: 496 (MH⁺)

H-R FAB/MS: Calcd. for C₂₆H₃₃N₅O₃S: 496.2382, Found: 496.2386

Example 31(E)-3-{2-[2-(Aminomethyl)morpholino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)-ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.9Hz), 2.84-3.14 (5H, m), 3.20 (2H,t, J=7.1Hz), 3.63-3.85 (3H, m), 3.85-3.92 (1H, m), 3.97-4.03 (1H, m),6.90 (1H, d, J=15.5Hz), 7.08 (1H, s), 7.25 (1H, d, J=7.3Hz), 7.33 (1H,s), 7.45 (1H, d, J=15.5Hz), 7.92 (2H, br), 8.82 (1H, d, J=7.6Hz)

FAB/MS; m/z: 484 (MH⁺)

H-R EI/MS: Calcd. for C₂₄H₂₉N₅O₄S: 484.2019, Found: 484.1999

Example 32(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-[(3R)-3-(methylamino)-tetrahydro-1H-1-pyrrolyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.8Hz), 2.11-2.31 (2H, m), 2.58-2.64(3H, m), 2.92-3.02 (1H, m), 3.13-3.21 (2H, m), 3.35-3.42 (2H, m),3.44-3.52 (1H, m), 3.65-3.75 (1H, m), 3.80-4.05 (3H, m), 6.86 (1H, d,J=15.2Hz), 7.11 (1H, s), 7.14 (1H, dd, J=7.3, 1.7Hz), 7.26 (1H, s), 7.68(1H, d, J=15.2Hz), 8.76 (1H, d, J=7.3Hz), 9.05-9.30 (1H, br)

FAB/MS; m/z: 468 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₉N₅O₃S: 468.2069, Found: 468.2085

Example 33((E)-3-{2-[(3S)-3-(Dimethylamino)tetrahydro-1H-1-pyrrolyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.8Hz), 1.68-1.80 (1H, m), 2.03-2.12(1H, m), 2.18 (6H, s), 2.64-2.73 (1H, m), 2.92-3.03 (1H, m), 3.10-3.18(2H, m), 2.49-2.58 (1H, m), 2.62-2.79 (3H, m), 6.78 (1H, d, J=15.1Hz),7.05-7.08 (2H, m), 7.22 (1H, s), 7.68 (1H, d, J=15.1Hz), 8.71 (1H, d,J=7.3Hz)

FAB/MS; m/z: 482 (MH⁺), 504 (M⁺+Na).

H-R FAB/MS: Calcd. for C₂₅H₃₁N₅O₃S: 482.2226, Found: 482.2231

Example 34(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(4-methyl-1,4-diazepan-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=6.8Hz), 2.08-2.10 (1H, m), 2.12-2.37(1H, m), 2.77 (3H, s), 2.92-3.03 (1H, m), 3.12-3.20 (2H, m), 3.50-3.72(5H, m), 3.75-3.87 (2H, m), 4.08-4.17 (1H, m), 6.80 (1H, d, J=15.4Hz),7.10 (1H, s), 7.16 (1H, dd, J=7.3, 1.2Hz), 7.28 (1H, s), 7.48 (1H, d,J=15.4Hz), 8.77 (1H, d, J=7.3Hz), 10.38 (1H, br)

FAB/MS; m/z: 482 (MH⁺), 504 (M⁺+Na)

H-R FAB/MS: Calcd. for C₂₅H₃₁N₅O₃S: 482.2226, Found: 482.2234

Example 35(E)-3-{2-(4-Aminopiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=7.1Hz), 1.58-1.72 (2H, m), 1.97-2.08(2H, m), 2.93-3.03 (1H, m), 3.06-3.17 (2H, m), 3.17-3.23 (2H, m),3.23-3.55 (3H, m), 3.60-3.75 (2H, m), 6.91 (1H, d, J=15.5Hz), 7.15 (1H,s), 7.21 (1H, d, J=7.1Hz), 7.34 (1H, s), 7.42 (1H, d, J=15.5Hz), 8.19(2H, br), 8.80 (1H, d, J=7.3Hz)

FAB/MS; m/z: 468 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₉N₅O₃S: 468.2069, Found: 468.2069

Example 36(E)-3-{2-[4-(Hydroxymethyl)piperidino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.9Hz), 1.12-1.35 (2H, m), 1.68-1.82(3H, m), 2.92-3.07 (3H, m), 3.12-3.21 (2H, m), 3.95-4.03 (2H, m),4.52-4.57 (1H, m), 6.87 (1H, d, J=15.5Hz), 7.07 (1H, s), 7.16 (1H, d,J=6.6Hz), 7.28 (1H, s), 7.41 (1H, d, J=15.5Hz), 8.75 (1H, d, J=7.1Hz)

FAB/MS; m/z: 483 (MH⁺), 505 (M⁺+Na)

H-R FAB/MS: Calcd. for C₂₅H₃₀N₄O₄S: 483.2066, Found: 483.2064

Example 37(E)-3-{2-(3-Aminopiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=6.8Hz), 1.58-1.72 (2H, m), 1.77-1.88(1H, m), 2.00-2.14 (1H, m), 2.92-3.05 (1H, m), 3.03-3.55 (8H, m), 6.91(1H, d, J=15.5Hz), 7.13 (1H, s), 7.24 (1H, d, J=6.8Hz), 7.37 (1H, s),7.45 (1H, d, J=15.5Hz), 8.25 (3H, br), 8.82 (1H, d, J=6.8Hz)

FAB/MS; m/z: 468 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₉N₅O₃S: 468.2069, Found: 468.2073

Example 38(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-[3-(methylamino)-piperidino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=6.8Hz), 1.55-1.78 (2H, m), 1.78-1.89(1H, m), 2.05-2.20 (1H, m), 2.62 (3H, s), 2.92-3.03 (1H, m), 3.05-3.55(8H, m), 3.98-4.07 (1H, m), 6.91 (1H, d, J=15.5Hz), 7.11 (1H, s), 7.24(1H, d, J=7.3Hz), 7.38 (1H, s), 7.44 (1H, d, J=15.5Hz), 8.82 (1H, d,J=7.3Hz), 8.90 (1H, br), 9.07 (1H, br)

EI/MS; m/z: 481 (M⁺)

FAB/MS; m/z: 482 (MH⁺)

H-R FAB/MS: Calcd. for C₂₅H₃₁N₅O₃S: 482.2226, Found: 482.2244

Example 39(E)-3-{2-[3-(Dimethylamino)piperidino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.8Hz), 1.52-1.80 (2H, m), 1.82-1.93(1H, m), 2.14-2.23 (1H, m), 2.49 (6H, s), 2.80-2.90 (1H, m), 2.90-3.01(2H, m), 3.01-3.12 (1H, m), 3.17-3.24 (2H, m), 3.32-3.40 (2H, m),4.02-4.12 (1H, m), 4.27-4.36 (1H, m), 6.74 (1H, s), 6.83 (1H, dd, J=7.3,1.7Hz), 7.08 (1H, d, J=15.6Hz), 7.19 (1H, s), 7.56 (1H, d, J=15.6Hz),8.85 (1H, d, J=7.3Hz)

EI/MS; m/z: 495 (M⁺)

FAB/MS; m/z: 496 (MH⁺)

H-R FAB/MS: Calcd. for C₂₆H₃₃N₅O₃S: 496.2382, Found: 496.2383

Example 40(E)-3-{2-[3-(Aminocarbonyl)piperidino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)-ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.9Hz), 1.55-1.67 (2H, m), 1.67-1.84(1H, m), 1.88-2.00 (1H, m), 2.40-2.50 (1H, m), 2.90-3.12 (3H, m),3.12-3.22 (2H, m), 3.81-3.91 (1H, m), 4.00-4.12 (1H, m), 6.86 (1H, d,J=15.5Hz), 6.89 (1H, s), 7.07 (1H, s), 7.17 (1H, d, J=7.1Hz), 7.29 (1H,br), 7.34 (1H, s), 7.41 (1H, d, J=15.5Hz), 8.76 (1H, d, J=7.1Hz), 11.89(1H, br)

FAB/MS; m/z: 496 (MH⁺)

H-R FAB/MS: Calcd. for C₂₅H₂₉N₅O₄S: 496.2019, Found: 496.2018

Example 41(E)-3-{2-[4-(Aminocarbonyl)piperidino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)-ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.9Hz), 1.60-1.76 (2H, m), 1.76-1.85(2H, m), 2.35-2.46 (1H, m), 2.92-3.09 (3H, m), 3.12-3.20 (2H, m),3.92-4.00 (2H, m), 6.82 (1H, s), 6.87 (1H, d, J=15.5Hz), 7.07 (1H, s),7.17 (1H, dd, J=7.3, 1.7Hz), 7.22-7.33 (2H, m), 7.42 (1H, d, J=15.5Hz),8.77 (1H, d, J=15.5Hz), 11.88 (1H, br)

FAB/MS; m/z: 496 (MH⁺)

H-R FAB/MS: Calcd. for C₂₅H₂₉N₅O₄S: 496.2019, Found: 496.2015

Example 42(E)-3-{2-[(7S)-7-Amino-5-azaspiro[2,4]hept-5-yl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

¹H-NMR (DMSO-d₆) δ: 1.70-1.90 (3H, m), 1.00-1.09 (1H, m), 1.20 (6H, d,J=6.9Hz), 2.91-3.02 (1H, m), 3.13-3.22 (2H, m), 3.22-3.28 (1H, m),3.62-3.75 (1H, m), 3.77-3.85 (1H, m), 4.19-4.32 (2H, m), 6.87 (1H, d,J=15.2Hz), 7.08 (1H, s), 7.14 (1H, d, J=6.9, 2.2Hz), 7.25 (1H, s), 7.68(1H, d, J=15.2Hz), 8.16 (2H, br), 8.75 (1H, d, J=6.9Hz)

FAB/MS; m/z: 480 (MH⁺)

H-R FAB/MS: Calcd. for C₂₅H₂₉N₅O₃S: 480.2069, Found: 480.2062

Example 43(E)-3-{2-[(3S,4S)-3-Amino-4-(fluoromethyl)tetrahydro-1H-1-pyrrolyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=6.9Hz), 2.83-3.07 (2H, m), 3.12-3.20(2H, m), 3.34-3.42 (2H, m), 3.65-3.83 (2H, m), 3.87-4.10 (3H, m),4.62-4.70 (1H, m), 4.72-4.81 (1H, m), 6.88 (1H, d, J=15.3Hz), 7.09 (1H,s), 7.14 (1H, dd, J=7.3, 1.7Hz), 7.27 (1H, s), 7.68 (1H, d, J=15.3Hz),8.29 (2H, br), 8.75 (1H, d, J=7.3Hz)

FAB/MS; m/z: 486 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₈FN₅O₃S: 486.1975, Found: 486.1974

Example 44(E)-3-{2-[(3R)-3-Hydroxypiperidino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.19 (6H, d, J=6.9Hz), 1.30-1.60 (2H, m), 1.73-1.83(1H, m), 1.83-1.96 (1H, m), 2.90-3.00 (2H, m), 3.08-3.21 (3H, m),3.52-3.61 (1H, m), 3.63-3.72 (1H, m), 3.82-3.91 (1H, m), 4.85-4.93 (1H,m), 6.84 (1H, d, J=15.4Hz), 7.07 (1H, s), 7.14 (1H, d, J=6.9Hz), 7.27(1H, s), 7.41 (1H, d, J=15.4Hz), 8.74 (1H, d, J=7.8Hz), 11.85 (1H, br).

FAB/MS; m/z: 469 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₈N₄O₄S: 469.1910, Found: 469.1901

Example 45(E)-3-{2-[(3S)-3-Hydroxypiperidino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)-ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.9Hz), 1.31-1.61 (2H, m), 1.74-1.85(1H, m), 1.85-1.98 (1H, m), 2.90-3.00 (2H, m), 3.08-3.21 (3H, m),3.53-3.63 (1H, m), 3.63-3.75 (1H, m), 3-83-3.92 (1H, m), 4.86-4.95 (1H,m), 6.85 (1H, d, J=15.7Hz), 7.07 (1H, s), 7.15 (1H, d, J=6.6Hz), 7.28(1H, s), 7.42 (1H, d, J=15.7Hz), 8.75 (1H, d, J=7.3Hz), 11.88 (1H, br)

FAB/MS; m/z: 469 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₈N₄O₄S: 469.1910, Found: 469.1921

Example 46(E)-3-{2-(3-Amino-1-azetanyl)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=6.8Hz), 2.93-3.03 (1H, m), 3.13-3.23(2H, m), 3.43-3.53 (2H, m), 3.65-3.85 (2H, m), 4.25-4.35 (2H, m),4.50-4.58 (1H, m), 6.92 (1H, d, J=15.2Hz), 7.10 (1H, s), 7.16 (1H, d,J=7.6Hz), 7.28 (1H, s), 7.50 (1H, d, J=15.2Hz), 8.49 (1H, br), 8.77 (1H,d, J=7.1Hz)

FAB/MS; m/z: 440 (MH⁺)

H-R FAB/MS: Calcd. for C₂₂H₂₅N₅O₃S: 440.1756, Found: 440.1768

Example 47(E)-3-{2-(4-Fluoropiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.8Hz), 1.75-1.88 (2H, m), 1.93-2.10(2H, m), 2.91-3.01 (1H, m), 3.14-3.21 (2H, m), 3.32-3.40 (2H, m),3.40-3.52 (2H, m), 3.52-3.68 (2H, m), 4.83-4.92 (0.5H, m), 4.95-5.04(0.5H, m), 6.88 (1H, d, J=15.6Hz), 7.07 (1H, s), 7.19 (1H, dd, J=7.1,1.5Hz), 7.33 (1H, s), 7.44 (1H, d, J=15.6Hz), 8.79 (1H, d, J=7.1Hz),11.91 (1H, br)

FAB/MS; m/z: 470 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₇FN₄O₃S: 470.1788, Found: 470.1779

Example 48(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-oxopiperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.8Hz), 2.91-3.01 (1H, m), 3.15-3.22(2H, m), 3.30-3.41 (6H, m), 3.79-3.87 (4H, m), 6.91 (1H, d, J=15.4Hz),7.07 (1H, s), 7.22 (1H, dd, J=7.3, 1.8Hz), 7.37 (1H, s), 7.52 (1H, d,J=15.4Hz), 8.81 (1H, d, J=7.3Hz), 11.94 (1H, br)

FAB/MS; m/z: 467 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₆N₄O₄S: 467.1753, Found: 467.1765

Example 49(E)-3-(8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-{4-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-2-pyranyl]-piperazino}-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.35 (6H, d, J=6.8Hz), 3.12-3.23 (2H, m), 3.25-3.43(4H, m), 3.53-3.90 (18H, m), 7.10 (1H, d, J=15.7Hz), 7.18 (1H, s), 7.35(1H, dd, J=7.1,2.4Hz), 7.42 (1H, s), 7.57 (1H, d, J=15.7Hz), 8.94 (1H,d, J=7.1Hz)

LC-MS; m/z: 616 (MH⁺)

Example 50(E)-3-[8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-{[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-2-pyranyl]oxy}-piperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoic acid

¹H-NMR (CD₃OD) δ: 1.26 (6H, d, J=6.8Hz), 1.70-1.92 (2H, m), 1.92-2.10(2H, m), 2.98-3.10 (1H, m), 3.17-3.25 (2H, m), 3.36-3.58 (7H, m),3.68-3.78 (2H, m), 3.82-3.98 (3H, m), 4.00-4.12 (1H, m), 4.39 (1H, d,J=7.3Hz), 6.95 (1H, d, J=15.6Hz), 6.96 (1H, s), 7.04 (1H, dd, J=7.3,1.7Hz), 7.21 (1H, s), 7.60 (1H, d, J=15.6Hz), 8.79 (1H, d, J=7.3Hz)

FAB/MS; m/z: 631 (MH⁺)

H-R FAB/MS: Calcd. for C₃₀H₃₈N₄O₉S: 631.2438, Found: 631.2485

Example 51(E)-3-{2-[cis-3,4-Dihydroxyhexahydro-1-pyridinyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.19 (6H, d, J=7.1Hz), 1.60-1.72 (1H, m), 1.77-1.88(1H, m), 2.90-3.01 (1H, m), 3.12-3.20 (2H, m), 3.35-3.42 (2H, m),3.45-3.66 (3H, m), 3.72-3.80 (1H, m), 4.57-4.60 (1H, m), 4.60-4.70 (1H,m), 6.83 (1H, d, J=15.5Hz), 7.06 (1H, s), 7.13 (1H, d, J=7.6Hz), 7.26(1H, s), 7.42 (1H, d, J=15.5Hz), 8.73 (1H, d, J=7.1Hz)

FAB/MS; m/z: 485 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₈N₄O₅S: 485.1859, Found: 485.1882

Example 523-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propanoicacid

(A) tert-Butyl3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-2-propanoate

tert-Butyl(E)-3-{8-[2-(4-isoprolyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenate(29.8 mg, 0.058 mmol) was dissolved in methanol (20 ml), added with 10%palladium/carbon (6.0 mg) and stirred at room temperature under hydrogenflow for 4 hours and 30 minutes. After the catalyst was removed byfiltration, the solvent was evaporated and the residue was purified bypreparative TLC (chloroform:methanol=30:1, v/v) to obtain the titlecompound (8.0 mg, 26.7%) as a pale yellow oily substance.

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.8Hz), 1.43 (9H, s), 2.62-2.70 (2H,m), 2.87-2.93 (2H, m), 3.02-3.12 (1H, m), 3.16-3.23 (2H, m), 3.32-3.47(6H, m), 3.80-3.86 (4H, m), 6.73 (1H, s), 6.82 (1H, dd, J=7.3, 1.7Hz),7.22 (1H, s), 8.81 (1H, d, J=7.3Hz)

(B)3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-2-propanoicacid

The tert-Butyl3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propanoate(8.0 mg, 0.016 mmol) obtained in (A) was added with 4 N hydrochloricacid in dioxane (1 ml), stirred at room temperature for 3 hours, furtheradded with 4 N hydrochloric acid in dioxane (1 ml), and stirred forfurther 4 hours. The solvent was evaporated, and the residue waspurified by preparative TLC (chloroform:methanol=10:1, v/v) andlyophilized from dioxane to obtain the title compound (4.3 mg, 60.4%) aspale yellow powder.

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.9Hz), 2.76 (2H, t, J=6.9Hz), 2.92(2H, t, J=6.9Hz), 3.02-3.12 (1H, m), 3.18-3.25 (2H, m), 3.3.4-3.56 (6H,m), 3.78-3.86 (4H, m), 6.73 (1H, s), 6.88 (1H, dd, J=7.3, 1.7Hz), 7.26(1H, s), 8.82 (1H, d, J=7.1Hz)

LC/MS; m/z: 457 (MH⁺), 455 (M⁺−1)

EI/MS; m/z: 456 (MH⁺)

H-R EI/MS: Calcd. for C₂₃H₂₈N₄O₄S: 456.1831, Found: 456.1848

Example 53(E)-3-{2-(4,4-Dimethylhexahydropyrazin-4-ium-1-yl)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(4-methyl)piperazino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid (19.9 mg, 0.043 mmol) was dissolved in dimethylformamide (1 ml),added dropwise with methyl iodide (0.1 ml, 1.61 mmol), sealed with astopper and left in a refrigerator for 14 hours. The solvent andexcessive reagents were evaporated, and the residue was lyophilized fromdioxane/water to obtain the title compound (29.7 mg, quantitative).

¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=6.9Hz), 2.55 (6H, s), 2.91-3.02 (1H,m), 3.15-3.26 (2H, m), 3.45-3.55 (4H, m), 3.82-3.92 (4H, m), 6.93 (1H,d, J=15.5Hz), 7.09 (1H, s), 7.29 (1H, d, J=7.1Hz), 7.41 (1H, s), 7.45(1H, d, J=15.5Hz), 8.14 (1H, br),8.85 (1H, d, J=7.3Hz)

FAB/MS; m/z: 482 (M⁺)

H-R FAB/MS: Calcd. for C₂₅H₃₂N₅O₃S: 482.2226, Found: 482.2216

Example 54(E)-3-{2-{(3R)-3-[(Aminocarbonyl)oxy]piperidino}-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-{2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenate

tert-Butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-[(4-methylphenyl)-sulfonyl]oxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenate(111.4 mg, 0.19 mmol) was dissolved in dimethylformamide (3 ml), addeddropwise with triethylamine (130.4 μl, 0.93 mmol), added withR-(+)-3-hydroxypiperidine hydrochloride (128.7 mg, 0.93 mmol), andstirred at room temperature for 17 hours. The solvent was evaporated,and then the residue was purified by preparative TLC(chloroform:methanol=30:1, v/v) to obtain the title compound (91.4 mg,93.2%) as yellow oil.

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.8Hz), 1.51 (9H, s), 1.72-1.95 (4H,m), 3.01-3.11 (1H, m), 3.16-3.23 (2H, m), 3.32-3.40 (2H, m), 3.49-3.68(3H, m), 3.88-3.97 (1H, m), 3.97-4.07 (1H, m), 6.74 (1H, s), 6.86 (1H,d, J=7.3Hz), 7.03 (1H, d, J=15.6Hz), 7.19 (1H, s), 7.49 (1H, d,J=15.6Hz), 8.85 (1H, d, J=7.3Hz)

(B) tert-Butyl(E)-3-{2-(3R)-3-[(aminocarbonyl)oxy]hexahydro-1-pyridinyl-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenate

The tert-Butyl(E)-3-{2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenate(38.9 mg, 0.074 mmol) obtained in (A) was dissolved in ethyl acetate (3ml), added with trichloroacetyl isocyanate (9.7 μl, 0.082 mmol) underice cooling, and stirred at the same temperature for 1 hour. Thereaction solution was further added with trichloroacetyl isocyanate (9.7μl, 0.082 mmol), stirred at the same temperature for further 1 hour, andadded with chloroform/methanol (10:1, v/v, 6 ml), and the solvent wasevaporated. The residue was dissolved in methanol (1.5 ml), added withwater (0.2 ml) and sodium formate (9.6 mg, 0.14 mmol) stirred at roomtemperature for 2 hours and 30 minutes. The mixture was further addedwith sodium formate (9.6 mg, 0.14 mmol), and stirred at room temperaturefor further 20 hours. The solvent was evaporated, and the residue waspurified by preparative TLC (chloroform:methanol=30:1, v/v) to obtainthe title compound (74.9 mg, quantitative) as yellow solid.

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.9Hz), 1.50 (9H, s), 1.80-2.10 (4H,m), 3.02-3.12 (1H, m), 3.15-3.23 (2H, m), 3.23-3.40 (3H, m), 3.45-3.63(1H, m), 3.65-3.77 (2H, m), 4.75-4.85 (1H, m), 6.73 (1H, s), 6.85 (1H,dd, J=7.3, 1.7Hz), 7.08 (1H, d, J=15.7Hz), 7.22 (1H, s), 7.73 (1H, d,J=15.7Hz), 8.86 (1H, d, J=7.1Hz)

(C)(E)-3-{2-{(3R)-3-[(Aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenicacid

The tert-Butyl(E)-3-{2-(3R)-3-[(aminocarbonyl)oxy]hexahydro-1-pyridinyl-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenate(74.9 mg, 0.074 mmol) obtained in (B) was dissolved in 4 N hydrochloricacid solution in dioxane and stirred at room temperature for 5 hours.After the solvent was evaporated, the residue was purified bypreparative TLC (chloroform:methanol=10:1, v/v) and lyophilized fromdioxane to obtain the title compound (17.6 mg, 67.5%) as yellow powder.

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.9Hz), 1.55-1.70 (2H, m), 1.80-1.90(1H, m), 1.90-2.02 (1H, m), 2.90-3.01 (1H, m), 3.13-3.20 (2H, m),3.30-3.42 (3H, m), 3.45-3.60 (2H, m), 3.80-3.88 (1H, m), 4.54-4.62 (1H,m), 6.49 (2H, br), 6.87 (1H, d, J=15.4Hz), 7.07 (1H, s,), 7.17 (1H, dd,J=7.3, 1.7Hz), 7.32 (1H, s), 7.44 (1H, d, J=15.4Hz), 8.76 (1H, d,J=7.3Hz), 11.89 (1H, br)

FAB/MS; m/z: 512 (MH⁺)

H-R FAB/MS: Calcd. for C₂₅H₂₉N₅O₅S: 512.1968, Found: 512.1970

Example 55(E)-3-{2-{(3S)-3-[(Aminocarbonyl)oxy]piperidino}-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The title compound was synthesized in the same manner as in Example 54.

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.9Hz), 1.53-1.70 (2H, m), 1.80-1.90(1H, m), 1.90-2.02 (1H, m), 2.91-3.01 (1H, m), 3.13-3.20 (2H, m),3.30-3.42 (3H, m), 3.45-3.62 (2H, m), 3.80-3.88 (1H, m), 4.53-4.63 (1H,m), 6.49 (2H, br), 6.87 (1H, d, J=15.4Hz), 7.07 (1H, s,), 7.17 (1H, dd,J=7.3, 1.7Hz), 7.32 (1H, s), 7.44 (1H, d, J=15.4Hz), 8.76 (1H, d,J=7.3Hz), 11.89 (1H, br)

FAB/MS; m/z: 512 (MH⁺)

H-R FAB/MS: Calcd. for C₂₅H₂₉N₅O₅S: 512.1968, Found: 512.1968

Example 56(E)-3-{2-{4-[(Aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The title compound was synthesized in the same manner as in Example 54.

¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J=6.9Hz), 1.57-1.70 (2H, m), 1.91-2.01(2H, m), 2.91-3.01 (1H, m), 3.13-3.20 (2H, m), 3.27-3.40 (4H, m),3.72-3.82 (2H, m), 4.69-4.78 (1H, m), 6.50 (2H, br), 6.86 (1H, d,J=15.4Hz), 7.07 (1H, s), 7.18 (1H, dd, J=7.3, 2.0Hz), 7.32 (1H, s), 7.44(1H, d, J=15.4Hz), 8.77 (1H.d, J=7.3Hz), 11.90 (1H, br)

FAB/MS; m/z: 512 (MH⁺)

H-R FAB/MS: Calcd. for C₂₅H₂₉N₅O₅S: 512.1968, Found: 512.1964

Example 578-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-3-[2-(2H-1,2,3,4-tetrazol-5-yl)acetyl]-4H-pyrido[1,2-a]pyrimidin-4-one

(A) Ethyl 2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]acetate

Ethyl 2-(1H-1,2,3,4-tetrazol-5-yl)acetate (5.0 g, 32.0 mmol) wasdissolved in dimethylformamide (20 ml), added with potassium carbonate(5.75 g, 41.6 mmol). The mixture was further added dropwise with4-methoxybenzyl chloride (5.21 ml, 38.4 mmol) under ice cooling andstirred at the same temperature for 1 hour and 30 minutes and at roomtemperature for 15 hours. The solvent was evaporated, and the residuewas diluted with toluene, washed with water and saturated brine, anddried over anhydrous sodium sulfate. Then, the solvent was evaporatedand the residue was purified by silica gel column chromatography(hexane:ethyl acetate=2:1, v/v) to obtain the title compound (3.78 g,42.7%) as colorless oil.

¹H-NMR (CDCl₃) δ: 1.25 (3H, t, J=7.1Hz), 3.79 (3H, s), 3.94 (2H, s),4.19 (2H,q, J=7.1Hz), 5.68 (2H, s), 6.82-6.92 (2H, m), 7.28-7.38 (2H, m)

(B) 2-[2-(4-Methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]acetic acid

The ethyl 2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]acetate (2.15g, 7.78 mmol) obtained in (A) was dissolved in tetrahydrofuran/methanol(3:1, v/v, 60 ml), added dropwise with a solution of lithium hydroxide(359.2 mg, 8.56 mmol) in water (15 ml) under ice cooling and thenstirred at room temperature for 2 hours. The solvent was evaporated, andthe residue was added with 1 N aqueous hydrochloric acid to obtain pH ofabout 1. The solution was extracted with ethyl acetate, and the organiclayer was washed with saturated brine and dried over anhydrous sodiumsulfate. Then, the solvent was evaporated to obtain the title compound(1.91 g, 98.8%).

¹H-NMR (CDCl₃) δ: 3.79 (3H, s), 3.99 (2H, s), 5.68 (2H, s),6.85-6.95(2H, m), 7.29-7.39 (21H, m)

(C) 2-[2-(4-Methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]ethanoyl chloride

The 2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]acetic acid (262.0mg, 1.05 mmol) obtained in (B) was added dropwise with thionyl chloride(615.9 μl , 8.44 mmol) under ice cooling and stirred at room temperaturefor 30 minutes. Excessive regents were evaporated to obtain the titlecompound (0.27 g, quantitative) as pale yellow oil.

(D)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-3-{2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]acetyl}-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one

The 2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]ethanoyl chloride(0.27 g, 1.05 mmol) obtained in (C) was dissolved in methylene chloride(3 ml), added dropwise with pyridine (170.2 μl, 2.10 mmol) under icecooling, and added dropwise with a solution of8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido-[1,2-a]pyrimidin-4-one(80.9 mg, 0.21 mmol) in methylene chloride (3 ml). The reaction solutionwas stirred at room temperature for 23 hours, then further added with asolution of 2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]ethanoylchloride (0.27 g, 1.05 mmol) in methylene chloride (2 ml) and pyridine(170.2 μl, 2.10 mmol) under ice cooling. Further, the reaction solutionwas added with the same amounts of the acid chloride and pyridine twiceevery 24 hours, and stirred at room temperature. The solvent wasevaporated, and the residue was added with saturated aqueous sodiumhydrogencarbonate and extracted with ethyl acetate. The organic layerwas washed with saturated brine and dried over anhydrous sodium sulfate.The solvent was evaporated and the residue was purified by preparativeTLC (hexane:ethyl acetate=1:1, v/v and chloroform:methanol=30:1, v/v) toobtain the title compound (10.1 mg, 7.8%) as yellow oil.

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.8Hz), 3.02-3.12 (1H, m), 3.12-3.20(2H, m), 3.31-3.38 (2H, m), 3.60-3.75 (8H, m), 3.79 (3H, s), 4.68 (2H,s), 5.66 (2H, s), 6.72 (1H, dd, J=7.3, 1.5Hz), 6.73 (1H, s), 6.86 (2H,d, J=8.6Hz), 7.03 (1H, s), 7.29 (2H, d, J=8.6Hz), 8.71 (1H, d, J=7.3Hz)

(E)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-3-[2-(2H-1,2,3,4-tetrazol-5-yl)acetyl]-4H-pyrido[1,2-a]pyrimidin-4-one

The8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-3-{2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-ylacetyl-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one(8.9 mg, 0.014 mmol) obtained in (D) was dissolved in trifluoroaceticacid (5 ml) and stirred at room temperature for 18 hours. The solventwas evaporated, and the residue was purified by preparative TLC(chloroform:methanol=10:1, v/v) and lyophilized from dioxane to obtainthe title compound (4.8 mg, 67.0%) as pale yellow powder.

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.9Hz), 3.01-3.12 (1H, m), 3.17-3.23(2H, m), 3.32-3.40 (2H, m), 3.55-3.63 (4H, m), 3.67-3.76 (4H, m), 4.75(2H, s), 6.74 (1H, s), 6.80 (1H, d, J=7.3Hz), 7.07 (1H, s), 8.73 (1H, d,J=7.3Hz)

FAB/MS; m/z: 495 (MH⁺)

H-R FAB/MS: Calcd. for C₂₃H₂₆N⁸O₃S: 495.1927, Found: 495.1955

Example 58(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3-[(dimethylamino)carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-{3-[(dimethylamino)carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide

N⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]-pyrimidine-8-carboxamide(301.9 mg, 0.88 mmol) was suspended in dimethylformamide (6 ml) andacetonitrile (12 ml), added dropwise with diisopropylethylamine (1.83ml, 10.5 mmol) and diphenyl chlorophosphate (545.1. μl, 2.63 mmol) at−10° C. under an argon flow, and stirred at the same temperature for 5minutes and at room temperature for 15 minutes. The reaction solutionwas cooled to −10° C. again, added dropwise with a solution of3-[(dimethylamino)carbonyl]piperidine trifluoroacetic acid salt (1.18 g,4.38 mmol) in dimethylformamide (5 ml), and stirred at room temperaturefor 1 hour and at about 80° C. for 2 hours. The reaction solution washeated to about 100° C., heated for 30 minutes with stirring, then addedwith diisopropylethylamine (1.83 ml, 10.5 mmol), and further heated at100° C. for 3 hours and 30 minutes with stirring. After cooling, thesolution was added with saturated aqueous sodium hydrogencarbonate andextracted with chloroform. The organic layer was washed with saturatedbrine and dried over anhydrous sodium sulfate and the solvent wasevaporated. The residue was purified by silica gel column chromatography(chloroform→chloroform:methanol=50:1 20:1, v/v) and preparative TLC(chloroform:methanol=20:1, v/v) to obtain the title compound (85.9 mg,20.3%) as yellow orange oil.

¹H-NMR (CDCl₃) δ: 1.33 (9H, s), 1.42-1.62 (1H, m), 1.70-2.08 (3H, m),2.57-3.20 (3H, m), 2.99 (3H, s), 3.12 (3H, s), 4.03-4.90 (2H, m), 5.69(1H, s), 6.60 (1H, s), 7.36-7.22 (1H, m), 7.89 (1H, s), 8.97 (1H, d,J=7.3Hz)

LC-MS; m/z: 483 (MH⁺)

(B)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-{3-[(dimethylamino)carbonyl]piperidino}-3-formyl-4-oxo-4H-pyrido[1,2-a]pyrimidine -8-carboxamide

Dimethylformamide (2 ml) was added dropwise with phosphorus oxychloride(24.9 μl, 0.27 mmol) under ice cooling, and stirred at room temperaturefor 30 minutes. The reaction solution was cooled with ice again, addeddropwise with a solution of theN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-{3-[(dimethylamino)carbonyl]-piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(85.9 mg, 0.18 mmol) obtained in (A) in dimethylformamide (2 ml) andstirred at the same temperature for 2 hours. The reaction solution wasadded with saturated aqueous sodium hydrogencarbonate and extracted withchloroform. The organic layer was washed with saturated brine and driedover anhydrous sodium sulfate, and the solvent was evaporated to obtainthe title compound (84.2 mg, quantitative) as yellow oil.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.57-2.10 (4H, m), 2.89 (3H, s), 2.97(3H, s), 3.00-3.28 (3H, m), 4.10-4.45 (2H, m), 6.59 (1H, s), 7.35-7.45(1H, m), 7.82 (1H, s), 8.95 (1H, s), 8.86 (1H, d, J=7.3Hz), 10.12 (1H,s)

ESI/MS; m/z: 511 (MH⁺)

(C) Methyl(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3-[(dimethylamino)carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

TheN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-{3-[(dimethylamino)carbonyl]-piperidino}-3-formyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(84.2 mg, 0.18 mmol) obtained in (B) was dissolved in tetrahydrofuran(10 ml), added with lithium chloride (45.3 mg, 1.07 mmol), and addeddropwise withbis(2,2,2-trifluoroethyl)-(methoxycarbonylmethyl)phosphonate (112.9 μl,0.53 mmol) and 1,8-diazabicyclo-[5,4,0]undec-7-ene (73.4 μl, 0.53 mmol).After the reaction solution was stirred at room temperature for 2 hours,the solvent was evaporated, and the residue was purified by preparativeTLC (chloroform:methanol=20:1, v/v) to obtain the title compound (63.7mg, 68.2%) as a mixture of orange oil and solid.

¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 1.56-1.70 (1H, m), 1.70-2.00 (3H, m),2.92-3.20 (3H, m), 2.99 (3H, s), 3.19 (3H, s), 3.78 (3H, s), 3.97-4.03(1H, m), 4.22-4.30 (1H, m), 6.60 (1H, s), 7.10 (1H, d, J=15.6Hz), 7.45(1H, dd, J=7.3, 1.7Hz), 7.49 (1H, d, J=15.6Hz), 7.85 (1H, d, J=1.7Hz),8.96 (1H, d, J=7.3Hz)

(D)(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3-[(dimethylamino)-carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

The methyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3-[(dimethylamino)carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate(63.7 mg, 0.11 mmol) obtained in (C) was dissolved in methanol (10 ml),added dropwise with 1 N aqueous sodium hydroxide (562.0 μl, 0.56 mmol),and stirred at room temperature for 1 hour. The reaction solution wasfurther added with 1 N aqueous sodium hydroxide (5.62 ml, 5.62 mmol),stirred at room temperature for 2 hours, further added with 1 N aqueoussodium hydroxide (2.81 ml, 2.81 mmol), and stirred at room temperaturefor 3 hours. The reaction solution was adjusted to about pH 2 with 1 Nhydrochloric acid aqueous solution and extracted withchloroform/methanol (10:1, v/v). The organic layer was washed withsaturated brine and dried over anhydrous sodium sulfate, and the solventwas evaporated. The residue was purified by preparative TLC(chloroform:methanol=20:1, v/v) and lyophilized from dioxane to obtainthe title compound (37.6 mg, 38.5%, for the three steps) as yelloworange powder.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.60-1.80 (3H, m), 1.85-1.94 (1H, m),2.84 (3H, s), 2.97-3.05 (1H, m), 3.12 (3H, s), 3.30-3.45 (2H, m),3.95-4.03 (1H, m), 4.08-4.15 (1H, m), 6.82-6.95 (1H, m), 6.93 (1H, d,J=15.6Hz), 7.44 (1H, d, J=15.6Hz), 7.60-7.67 (1H, m), 8.12-8.18 (1H, m),8.90 (1H, d, J=7.3Hz)

FAB/MS; m/z: 553 (MH⁺)

H-R FAB/MS: Calcd. for C₂₇H₃₂N₆O₅S: 553.2233, Found: 553.2236

Example 59(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-piperidino-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

The title compound was synthesized in the same manner as in Example 58.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.62-1.72 (6H, m), 3.53-3.62 (4H, m),6.82-6.92 (1H, m), 6.93 (1H, d, J=15.6Hz), 7.45 (1H, d, J=15.6Hz),7.57-7.62 (1H, m), 8.14-8.20 (1H, m), 8.89 (1H, d, J=7.3Hz)

FAB/MS; m/z: 482 (MH⁺)

H-R FAB/MS: Calcd. for C₂₄H₂₇N₅O₄S: 482.1862, Found: 482.1844

Example 60(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3-[(methylamino)carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoic acid

The title compound was synthesized in the same manner as in Example 58.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.58-1.80 (3H, m), 1.89-1.97 (1H, m),2.59 (3H, d, J=4.4Hz), 3.18-3.20 (2H, m), 3.30-3.42 (2H, m), 3.90-3.97(1H, m), 4.11-4.18 (1H, m), 6.79-6.89 (1H, m), 6.93 (1H, d, J=15.6Hz),7.43 (1H, d, J=15.6Hz), 7.60-7.66 (1H, m), 7.77-7.83 (1H, m), 8.19-8.26(1H, m), 8.90 (1H, d, J=7.6Hz)

FAB/MS; m/z: 539 (MH⁺)

H-R FAB/MS: Calcd. for C₂₆H₃₀N₆O₅S: 539.2077, Found: 539.2112

Example 61(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) 2-Hydroxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one

2-Amino-4-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-pyridine (9.8 g) andbis(2,4,6-trichlorophenyl) malonate (18 g) were refluxed by heating inxylene for 30 minutes and left stand for cooling. The reaction mixturewas added with ether, and the deposited crystals were collected byfiltration, washed with ethyl acetate and dried to obtain the titlecompound (10.3 g). The reaction solution was combined and the solventwas evaporated. The residue was purified by silica gel columnchromatography to further obtain the title compound (1.5 g).

¹H-NMR (CDCl₃): 1.29 (6H, d, J=6.8Hz), 3.06 (1H, m), 3.34 (2H, m), 3.38(2H, m), 5.34 (1H, s), 6.74 (1H, s), 7.10 (1H, dd, J=7.1,1.7Hz), 7.37(1H, s), 9.02 (1H, d, J=7.1Hz)

(B)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one

The2-hydroxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]-pyrimidin-4-one(500 mg) obtained in (A) was dissolved in methylene chloride (20 ml),added with triethylamine (0.26 ml) and p-toluenesulfonyl chloride (360mg) and stirred for 24 hours under nitrogen atmosphere. The reactionsolution was added with morpholine (0.83 ml) and stirred for 12 hours,and the solvent was evaporated. The residue was purified by silica gelcolumn chromatography to obtain the title compound (273 mg).

¹H-NMR (CDCl₃): 1.30 (s, 3H), 1.33 (s, 3H), 3.07 (m, 1H), 3.18 (m, 2H),3.48 (m, 2H), 3.67 (m, 4H), 3.79 (m, 4H), 5.59 (s, 1H), 6.77 (s, 1H),6.78 (d, 1H), 7.11 (s, 1H), 8.80 (d, 1H)

(C)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]-pyrimidine-3-carbaldehyde

Dimethylformamide (10 ml) was added with phosphorus oxychloride (0.60ml) under ice cooling, stirred for 30 minutes, then added with the8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one(1.0 g) obtained in (B), stirred for 1 hour, and added with saturatedaqueous sodium hydrogencarbonate to adjust the reaction solution to pHof about 8. The reaction solution was extracted with ethyl acetate, andthe organic layer was washed with saturated brine and dried over sodiumsulfate. The solvent was evaporated under reduced pressure to obtain thetitle compound (1.07 g).

¹H-NMR (CDCl₃): 1.31 (s, 3H), 1.33 (s, 3H), 3.10 (m, 1H), 3.22 (m, 2H),3.42 (m, 2H), 3.73 (m, 4H), 3.81 (m, 4H), 6.80 (s, 1H), 6.82 (d, 1H),7.09 (s, 1H), 8.75 (d, 1H), 10.11 (s, 1H)

(D) tert-Butyl(E)-3-(8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

The8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde(110 mg) obtained in (C) and(tert-butoxycarbonylmethylene)triphenylphosphorane (441 mg) were stirredin tetrahydrofuran (5 ml) at 80° C. for 15 hours. After the solvent wasevaporated, the residue was purified by silica gel column chromatographyto obtain the title compound (150 mg) as yellow powder.

¹H-NMR (CDCl₃): 1.29 (6H, d, J=6.8Hz), 1.51 (9H, s), 3.05 (1H, m), 3.20(2H, m), 3.37 (2H, m), 3.60 (4H, m), 3.83 (4H, m), 6.73 (1H, s), 6.85(1H, d, J=6.8Hz), 7.05 (1H, d, J=15.4Hz), 7.20 (1H, s), 7.50 (1H, d,J=15.4Hz), 8.87 (1H, d, J=7.1Hz)

(E)(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(39 mg) obtained in (D) was stirred in formic acid (1 ml) for 4.5 hours,and the solvent was evaporated under reduced pressure to obtain thetitle compound (30 mg).

¹H-NMR (CD₃OD): 1.26 (6H, d, J=6.8Hz), 3.03 (1H, m), 3.20 (2H, m), 3.31(2H, m), 3.56 (4H, m), 3.79 (4H, m), 6.94 (1H, d, J=15.6Hz), 6.96 (1H,s), 7.05 (1H, d, J=6.6Hz), 7.21 (1H, s), 7.55 (1H, d, J=15.6Hz), 8.76(1H, d, J=7.1Hz)

The compounds of Examples 62 to 77 mentioned below were synthesized inthe same manner as in Example 61.

Example 62(E)-3-(2-Morpholino-4-oxo-8-{2-[4-(trifluoromethyl)-1,3-thiazol-2-yl]-ethyl}-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

¹H-NMR (CD₃OD) δ: 3.26 (m, 2H), 3.50 (m, 2H), 3.61 (m, 4H), 3.81 (m,4H), 6.68 (d, J=15.6Hz, 1H), 7.12 (d, 1H), 7.32 (s, 1H), 7.62 (d,J=16Hz, 1H), 8.03 (s, 1H), 8.84 (d, 1H)

MS (ES+) m/z 481 (M⁺+1)

Example 63(E)-3-{8-[2-(4-tert-Butyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.31 (s, 9H), 3.19 (t, 2H), 3.35 (t, 2H), 3.59 (m,4H), 3.79 (m, 4H), 6.72 (s, 1H), 6.85 (d, 1H), 7.07 (d, J=16Hz, 1H),7.18 (s, 1H), 7.62 (d, J=16Hz, 1H), 8.84 (d, 1H)

MS (ES+) m/z 469 (M⁺+1); MS (ES-) m/z 467 (M⁺−1)

Example 64(E)-3-{8-(2-[4-(1-Methylcyclopropyl)-1,3-thiazol-2-yl]ethyl)-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 0.70 (m, 2H), 1.03 (m, 2H), 1.41 (s, 3H), 3.18 (t,2H), 3.37 (t, 2H), 3.59 (m, 4H), 3.80 (m, 4H), 6.92 (s, 1H), 6.98 (d,J=16Hz, 1H), 7.08 (s, 1H), 7.25 (s, 1H), 7.60 (d, J=16Hz, 1H), 8.82 (d,1H)

MS (ES+) m/z 467 (M⁺+1); MS (ES−) m/z 465 (M⁺−1)

Example 65(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-carboxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.26 (d, 6H), 2.38 (m, 1H), 2.55 (m, 1H), 2.67 (m,1H), 2.82 (m, 1H), 3.08 (m, 1H), 3.12 (m, 2H), 3.39 (m, 3H), 3.56 (m,2H), 3.82 (m, 2H), 3.92 (m, 1H), 6.75 (s, 1H), 6.84 (d, 1H), 7.07 (d,J=14Hz, 1H), 7.21 (s, 1H), 7.33 (s, 1H), 7.20 (s, 1H), 7.65 (m, 2H),8.82 (d, 1H)

MS (ES+) m/z 497 (M⁺+1); MS (ES−) m/z 495 (M⁺−1)

Example 66(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(4-carboxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.29 (d, 6H), 2.00 (m, 2H), 2.13 (m, 2H), 2.62 (m,1H), 3.10 (m, 3H), 3.20 (m, 2H), 3.39 (m, 2H), 4.08 (m, 2H), 6.75 (s,1H), 6.83 (d, 1H), 7.08 (d, J=15.6Hz, 1H), 7.22 (s, 1H), 7.69 (d,J=15.6Hz, 1H), 8.86 (d, 1H)

MS (ES+) m/z 497 (M⁺+1); MS (ES−) m/z 495 (M⁺−1)

Example 67(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(1,2,3,4-tetrahydro-2-isoquinolinyl)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.33 (d, 6H), 2.95 (m, 1H), 3.12 (m, 2H), 3.23 (m,2H), 3.56 (t, 2H), 3.88 (t, 2H), 4.81 (s, 2H), 6.88 (m, 2H), 7.11 (d,J=15Hz, 1H), 7.19 (m, 6H), 7.79 (d, J=15Hz, 1H), 8.86 (d, 1H)

MS (ES+) m/z 501 (M⁺+1)

Example 68(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-hydroxy-3-methylpiperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.12 (s, 3H), 1.23 (d, 6H), 1.65 (m, 2H), 1.97 (m,1H), 3.08 (m, 1H), 3.2 (m, 3H), 3.39 (m, 4H), 3.68 (m, 2H), 6.95 (d,J=14Hz, 1H), 6.98 (s, 1H), 7.02 (d, 1H), 7.21 (s, 1H), 7.62 (d, J=14Hz,1H), 8.78 (d, 1H)

MS (ES+) m/z 483 (M⁺+1); MS (ES−) m/z 481 (M⁺−1)

Example 69(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-cyanopiperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.30 (d, 6H), 1.65 (m, 1H), 1.90 (m, 2H), 2.00 (m,1H), 2.98 (m, 1H), 3.08 (m, 1H), 3.22 (m, 2H), 3.40 (m, 2H), 3.52 (m,1H), 3.65 (m, 1H), 3.83 (m, 1H), 4.18 (m, 1H), 6.75 (s, 1H), 6.92 (d,1H), 7.11 (d, J=15.6Hz, 1H), 7.25 (s, with CDCl₃, 1H), 7.63 (d,J=15.6Hz, 1H), 8.88 (d, 1H)

MS (ES+) m/z 478 (M⁺+1); MS (ES−) m/z 476 (M⁺−1)

Example 70(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-cyanopiperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.33 (d, 6H), 2.04 (m, 4H), 2.95 (m, 1H), 3.21 (m,3H), 3.55 (m, 4H), 3.79 (m, 2H), 6.92 (m, 2H), 7.08 (d, J=15.6Hz, 1H),7.26 (1Hs, with CHCl₃, 1H), 7.62 (d, J=15.6Hz, 1H), 8.89 (d, 1H)

MS (ES+) m/z 478 (M⁺+1)

Example 71(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-cyanomorpholino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.29 (d, 6H), 3.05 (m, 1H), 3.22 (t, 2H), 3.40 (t,2H), 3.46 (m, 1H), 3.7-4.0 (m, 4H), 4.08 (m, 1H), 4.72 (m, 1H), 6.74 (s,1H), 6.97 (d, 1H), 7.14.(d, J=15.6Hz, 1H), 7.28 (s, 1H), 7.67 (d,J=15.6Hz, 1H), 8.90 (d, 1H) MS (ES+) m/z 480 (M⁺+1); MS (ES−) m/z 478(M⁺−1)

Example 72(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-aminocarbonylpiperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.25 (d, 6H), 3.02 (m, 1H), 3.2-3.4 (m, with CHD₂OD),4.24 (m, 1H), 6.96 (s, 1H), 7.04 (d, J=16Hz, 1H), 7.14 (m, 1H), 7.37 (s,1H), 7.58 (d, J=16Hz, 1H), 8.84 (d, 1H)

MS (ES+) m/z 497 (M⁺+1)

Example 73(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-carboxypiperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

¹H-NMR (CD₃OD) δ: 1.23 (d, 6H), 3.02 (m, 1H), 3.15-3.42 (m, withCHD₂OD), 3.62 (m, 1H), 3.95 (m, 1H), 4.75 (m, 1H), 6.96 (s, 1H), 7.05(m, 2H), 7.37 (m, 2H), 8.82 (d, 1H)

MS (ES+) m/z 520 (M⁺+Na); MS (ES−) m/z 496 (M⁺−1)

Example 74(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-cyanopiperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (300MHz, CD₃OD) δ: 1.23 (d, 6H), 3.02 (m, 1H), 3.15-3.42 (m, withCHD₂OD), 3.71 (m, 1H), 3.92 (m, 1H), 4.12 (m, 1H), 6.81 (s, 1H), 7.00(m, 2H), 7.25 (m, 1H), 7.52 (d, J=16Hz, 1H), 8.82 (d, 1H)

Example 75(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-carboxymorpholino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃+CD₃OD) δ: 1.22 (d, 6H), 3.0 (m, 1H), 3.14 (t, 2H), 3.31(m, 4H), 3.73 (m, 2H), 4.02 (m, 1H), 4.12 (m, 2H), 6.70 (s, 1H), 6.88(d, 1H), 6.97 (d, J=15.6Hz, 1H), 7.21 (s, 1H), 7.53 (d, J=15.6Hz, 1H),8.78 (d, 1H).

MS (ES+) m/z 499 (M⁺+1)

Example 76(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-aminocarbonylmorpholino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.30 (d, 6H), 3.0-3.2 (m, 5H), 3.47 (m, 2H), 3.81 (m,2H), 4.02 (m, 1H), 4.21 (m, 1H), 4.42 (m, 1H), 6.82 (s, 1H), 6.92 (d,1H), 7.08 (d, J=15.6Hz, 1H), 7.26 (s, with CHCl_(3,) 1H), 7.64 (d,J=15.6Hz, 1H), 8.87 (d, 1H)

MS (ES+) m/z 498 (M⁺+1); MS (ES−) m/z 496 (M⁺−1)

Example 77(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl-4-oxo-2-([(2S,3R,4R,5S,6S)-3,4,5,6-tetrahydroxytetrahydro-2H-2-pyranyl]methylamino)-4H-pyrido[1,2-a]-pyrimidin-3-yl]-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.23 (d, 6H), 3.02 (m, 1H), 3.18 (m, 2H), 3.4 (m, 3H),3.75 (m, 1H), 3.98 (m, 1H), 4.5 & 5.12 (2 doublets, mixture=of anomers,1H), 6.98 (s, 1H), 7.02 (m, 2H), 7.18 (s, 1H), 7.73 (d, J=16Hz, 1H),8.75 (d, 1H)

MS (ES+) m/z 547 (M⁺+1); MS (ES−) m/z 545 (M⁺−1)

Example 78(E)-3-{2-[4-((2S)-2-Amino-5-([amino(imino)methyl]amino}pentanoyl)-piperazino]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-2-propenoicacid

tert-Butyl(E)-3-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-2-piperazino-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(10 mg, 0.0197 mmol) and N-α, ω-1,ω-2-tri-tert-butoxycarbonyl-L-arginine (BOC-Arg, (BOC)₂OH, 14 mg, 0.0295mmol) were added with methylene chloride (1 ml). The mixture was furtheradded with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC HCl, 11 mg, 0.0591 mmol) and stirred at room temperature overnight.The reaction solution was diluted with methylene chloride, washed with5% aqueous citric acid, saturated aqueous sodium hydrogencarbonate andsaturated brine, and dried over sodium sulfate. The solvent wasevaporated under reduced pressure, and then the residue was added withtrifluoroacetic acid (1 ml) and stirred in the dark for 1 hour. Thereaction solution was added with toluene, and the solvent was evaporatedunder reduced pressure. The residue was purified by medium pressurecolumn chromatography (Amberkron column, gradient 100% 0.1% TFA in H₂Oto 100% CH₃CN over 80 minutes, 3 ml/minute) to obtain the title compound(7.0 mg).

¹H-NMR (CD₃OD): 1.34 (d, 6H), 1.70 (m, 2H), 1.90 (m, 2H), 3.10 (m, 1H),3.70 (m, 8H), 3.92 (m, 1H), 4.54 (t, 1H), 7.08 (d, 1H), 7.28 (s, 1H),7.52 (d, 1H), 7.55 (m, 2H), 7.66 (d, 1H), 7.72 (d, 1H), 8.93 (d, 1H)

MS (ES+): 631 (M+Na)

The compounds of Examples 79 to 91 mentioned below were synthesized inthe same manner as in Example 78.

Example 79(E)-3-{2-[4-((2S)-2-Amino-5-{[amino(imino)methyl]amino}pentanoyl)piperazino]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD): 1.26 (d, 6H), 1.70 (m, 2H), 1.90 (m, 2H), 3.03 (m, 1H),3.20-3.42 (m, 7H), 3.50-3.80 (m, 8H), 3.90 (m, 1H), 4.53 (t, 1H), 6.98(s, 1H), 7.05 (d, 1H), 7.15 (d, 1H), 7.30 (s, 1H), 7.64 (d, 1H), 8.87(d, 1H)

MS (ES+): 610

Example 80 (E)-3-{2-[4-((2S)-2-Amino-5-{[amino(imino)methyl]amino}pentanoyl)-piperazino]-8-[(E)-2-(4-tert-butyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD): 1.29 (s, 9H), 1.69 (m, 2H), 1.88 (m, 2H), 3.10-3.50 (m,5H), 3.50-3.80 (m, 10H), 3.90 (m, 2H), 4.53 (m, 1H), 6.96 (s, 1H), 7.05(d, 1H), 7.16 (d, 1H), 7.30 (s, 1H), 7.65 (d, 1H), 8.88 (d, 1H)

MS (ES+): 624

Example 81(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-dimethylaminoethylaminocarbonylpiperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.26 (d, J=7Hz, 6H), 1.80 (m, 4H), 2.05 (m, 1H), 2.65(m, 1H), 2.94 (s, 6H), 3.05 (m, 2H), 3.15-3.25 (m, 4H), 3.42 (t, 2H),3.58 (t, 2H), 4.10 (m, 2H), 6.98 (d, J=15.6Hz, 1H), 7.03 (s, 1H), 7.06(dd, 1H), 7.23 (s, 1H), 7.58 (d, J=15.6Hz, 1H), 8.80 (d, 1H)

MS (ES+) m/z 567 (M⁺+1)

Example 82(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-dimethylaminoethylaminocarbonylpiperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.26 (d, J=7Hz, 6H), 1.88 (m, 4H), 2.53 (m, 1H), 2.94(s, 6H), 3.02 (m, 1H), 3.0-3.25 (m, 6H), 3.41 (t, 2H), 3.55 (t, 2H),4.15 (m, 2H), 6.97 (m, 2H), 7.04 (dd, 1H), 7.22 (s, 1H), 7.60 (d,J=15.6Hz, 1H), 8.80 (d, 1H)

MS (ES+) m/z 567 (M⁺+1)

Example 83(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-dimethylaminoacetylpiperazino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.23 (d, 6H), 2.95 (s, 6H), 3.02 (m, 1H), 3.2-3.35 (m,with CHD₂OD), 3.42 (m, 1H), 3.58 (m, 1H), 3.63 (m, 2H), 3.80 (m, 1H),4.28 (s, 2H), 6.96 (s, 1H), 7.05 (d, J=13Hz, 1H), 7.14 (d, 1H), 7.29 (s,1H), 7.62 (d, J=13Hz, 1H), 8.85 (d, 1H)

Example 84(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(aminoethylthioethylamino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

¹H-NMR (CD₃OD) δ: 1.26 (d, J=7Hz, 6H), 2.85 (m, 4H), 3.02 (m, 1H), 3.2(m, 4H), 3.40 (t, 2H), 3.78 (t, 2H), 6.99 (m, 2H), 7.11 (d, J=15.6Hz,1H), 7.15 (s, 1H), 7.73 (d, J=15.6Hz, 1H), 8.78 (d, 1H)

MS (ES+) m/z 488 (M⁺+1)

Example 85(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-aminopropylaminocarbonylpiperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.26 (d, J=7Hz, 6H), 1.84 (m, 3H), 2.06 (m, 1H), 2.68(m, 1H), 2.93 (m, 2H), 3.02 (m, 2H), 3.2-3.3 (m, with CD₃OD), 3.42 (m,3H), 4.08 (m, 2H), 6.9-7.02 (m, 2H), 7.07 (dd, 1H), 7.25 (s, 1H), 7.58(d, J=15.6Hz, 1H), 8.81 (d, 1H)

MS (ES+) m/z 553 (M⁺+1)

Example 86(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-aminoethylaminocarbonylpiperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.26 (d, J=7Hz, 6H), 1.79 (m, 3H), 2.06 (m, 1H), 2.68(m, 1H), 3.0-3.1 (m, 4H), 3.15-3.28 (m, 3H), 3.45 (m, 4H), 4.1 (m, 2H),6.97 (d, J=15.6Hz, 1H), 7.06 (m, 2H), 7.25 (s, 1H), 7.58 (d, J=15.6Hz,1H), 8.79 (d, 1H)

MS (ES+) m/z 539 (M⁺+1)

Example 87(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-[(tetrahydro-1H-2-pyrrolylmethyl)amino]carbonylpiperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.26 (d, J=7Hz, 6H), 1.79 (m, 4H), 2.04 (m, 4H), 2.68(m, 1H), 2.9-3.1 (m, 2H), 3.15-3.35 (m, with CD₃OD), 3.35-3.55 (m, 4H),3.68 (m, 1H), 4.1 (m, 2H), 6.99 (m, 2H), 7.08 (d, 1H), 7.25 (s, 1H),7.59 (d, J=15.6Hz, 1H), 8.81 (d, 1H)

MS (ES+) m/z 579 (M⁺+1)

Example 88(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-aminomethylcarbonylpiperazino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.26 (d, J=7Hz, 6H), 3.02 (m, 1H), 3.23 (t, 2H), 3.42(t, 2H), 3.62 (m, 6H), 3.78 (m, 2H), 3.99 (s, 2H), 6.98 (s, 1H), 7.02(d, J=15.6Hz, 1H), 7.13 (d, 1H), 7.27 (s, 1H), 7.63 (d, J=15.6Hz, 1H),8.84 (d, 1H)

MS (ES+) m/z 511 (M⁺+1)

Example 89(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-prolylpiperazino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

¹H-NMR (CD₃OD) δ: 1.26 (d, J=7Hz, 6H), 1.9-2.1 (m, 3H), 2.52 (m, 1H),3.02 (m, 1H), 3.25 (t, 2H), 3.42 (t, 2H), 3.5-3.9 (m, 10H), 4.68 (m,1H), 6.97 (s, 1H), 7.03 (d, J=15.6Hz, 1H), 7.14 (d, 1H), 7.28 (s, 1H),7.63 (d, J=15.6Hz, 1H), 8.83 (d, 1H, d)

MS (ES+) m/z 551 (M⁺+1)

Example 90(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-lysylpiperazino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.26 (d, J=7Hz, 6H), 1.51 (m, 2H), 1.72 (m, 2H), 1.90(m, 2H), 2.95 (m, 2H), 3.02 (m, 1H), 3.25 (t, 2H), 3.42 (t, 2H), 3.5-3.8(m, 7H), 3.95 (m, 1H), 4.50 (m, 1H), 6.98 (s, 1H), 7.04 (d, J=15.6Hz,1H), 7.14 (d, 1H), 7.28 (s, 1H), 7.64 (d, J=15.6Hz, 1H), 8.85 (d, 1H)

MS (ES+) m/z 582 (M⁺+1)

Example 91(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-ornithylpiperazino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

¹H-NMR (CD₃OD) δ: 1.26 (d, J=7Hz, 6H), 1.80 (m, 2H), 1.92 (m, 2H), 2.99(m, 3H), 3.22 (t, 2H), 3.42 (t, 2H), 3.5-3.8 (m, 7H), 3.94 (m, 1H), 4.58(m, 1H), 6.99 (s, 1H), 7.04 (d, J=15.6Hz, 1H), 7.14 (d, 1H), 7.28 (s,1H), 7.64 (d, J=15.6Hz, 1H), 8.85 (d, 1H)

MS (ES+) m/z 568 (M⁺+1)

Example 92(E)-3-{2-(4-[2-(4-Aza-1-azoniabicyclo[2.2.2]oct-1-yl)acetyl]piperazino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-{2-[4-(2-chloroacetyl)piperazino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl(E)-3-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-piperazino-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(206 mg, 0.405 mmol) was dissolved in methylene chloride (8 ml), addedwith triethylamine (170 ml) and chloroacetyl chloride (64 ml) at −78°C., and then stirred overnight at room temperature in the dark. Thereaction solution was diluted with methylene chloride, washed withwater, 5% aqueous citric acid, saturated aqueous sodiumhydrogencarbonate and then with saturated brine, and the solution wasdried over magnesium sulfate. Then, the solvent was evaporated underreduced pressure. The residue was purified by silica gel columnchromatography to obtain the title compound (260 mg).

¹H-NMR (CDCl₃): 1.28 (d, 6H), 1.51 (s, 9H), 3.06 (m, 1H), 3.21 (m, 2H),3.37 (m, 2H), 3.62 (brm, 6H), 3.78 (brm, 2H), 4.10 (s, 2H), 6.72 (s,1H), 6.89 (d, 1H), 7.07 (d, 1H), 7.24 (d, 1H), 7.49 (d, 1H), 8.88 (d,1H)

(B) tert-Butyl(E)-3-{2-[4-(2-iodoacetyl)piperazino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)-ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

The tert-butyl(E)-3-{2-[4-(2-chloroacetyl)piperazino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(240 mg, 0.405 mmol) obtained in (A) and NaI (300 mg, 2.03 mmol) wererefluxed by heating in acetone (8 ml) for 3 hours in the dark. Thereaction solution was cooled, then diluted with methylene chloride,washed with water and saturated brine, and dried over sodium sulfate.Then, the solvent was evaporated under reduced pressure to obtain thetitle compound (226 mg) as a yellow amorphous substance.

¹H-NMR (CDCl₃): 1.28 (d, 6H), 1.52 (s, 9H), 3.10 (m, 1H), 3.22 (m, 2H),3.38 (m, 2H), 3.60 (m, 4H), 3.79 (s, 2H), 6.73 (s, 1H), 6.90 (d, 1H),7.08 (d, 1H), 7.50 (d, 1H), 8.88 (d, 1H)

(C)(E)-3-{2-(4-[2-(4-Aza-1-azoniabicyclo[2.2.2]oct-1-yl)acetyl]piperazino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl-1-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-butyl(E)-3-{2-[4-(2-iodoacetyl)piperazino]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(226 mg, 0.33 mmol) obtained in (B) and 1,4-diazabicyclo[2.2.2]octane(187 mg, 1.67 mmol) were stirred overnight in tetrahydrofuran (9 ml)under nitrogen atmosphere, then the solvent was evaporated, and theresidue was stirred in trifluoroacetic acid (TFA, 4 ml) for 1 hour. Thesolvent was evaporated under reduced pressure, and the residue waspurified by medium pressure reverse phase chromatography (Amberkroncolumn, gradient 100% 0.1% TFA in H₂O to 100% CH₃CN over 80 minutes, 3ml/minute) to obtain the title compound (150 mg, 75%, for the twosteps).

¹H-NMR (CD₃OD) δ: 1.27 (d, 6H), 3.02 (m, 1H), 3.26 (t, 2H), 3.43 (t,2H), 3.62 (m, 5H), 3.78 (m, 6H), 4.43 (s, 2H), 7.00 (s, 1H), 7.02 (d,J=15.6Hz, 1H), 7.13 (d, J=7Hz, 1H), 7.29 (s, 1H), 7.63 (d, J=15.6Hz,1H), 8.85 (d, J=7Hz, 1H)

MS (ES+) m/z 606 (M⁺+1)

The compounds of Examples 93 to 97 mentioned below were synthesized inthe same manner as in Example 92.

Example 93(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(4-[2-(1-methyl-1H-imidazol-3-ium-3-yl)acetyl]piperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

¹H-NMR (CDCl₃+CD₃OD) δ: 1.23 (d, J=7Hz, 6H), 3.02 (m, 1H), 3.17 (t, 2H),3.34 (t, 2H), 3.57 (m, 2H), 3.6-3.8 (m, 6H), 3.92 (s, 3H), 5.40 (s, 2H),6.72 (s, 1H), 6.88 (d, J=7.2Hz, 1H), 7.00 (d, J=15.6Hz, 1H), 7.20 (m,2H), 7.37 (s, 1H), 7.55 (d, J=15.6Hz, 1H), 8.81 (d, J=7.2Hz, 1H), 9.24(s, 1H)

MS (ES+) m/z 576 (M⁺+1)

Example 94(E)-3-{2-4-[2-(1-Azabicyclo[2.2.2]oct-1-yl)acetyl]piperazino-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD): 1.26 (d, 6H), 2.05 (brm, 7H), 2.19 (m, 1H), 3.03 (m,1H), 3.22 (m, 2H), 3.39 (m, 2H), 3.61 (brm, 6H), 3.67 (m, 8H), 4.26 (s,2H), 6.97 (s, 1H), 6.99 (d, 1H), 7.12 (d, 1H), 7.28 (s, 1H), 7.62 (d,1H), 8.85 (d, 1H)

MS (ES+): 605

Example 95(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-[2-(1-pyridinium)acetyl]piperazino)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD): 1.27 (d, 6H), 3.02 (m, 1H), 3.25 (m, 2H), 3.40 (m, 2H),5.80 (s, 2H), 6.98 (s, 11H), 7.02 (d, 1H), 7.17 (d, 1H), 7.31 (s, 1H),7.65 (d, 1H), 8.20 (m, 2H), 8.65 (m, 1H), 8.88 (m, 2H)

MS (ES+): 573

Example 96(E)-3-{2-(4-[2-(1-Azabicyclo[2.2.2]oct-1-yl)butanoyl]piperazino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD): 1.25 (d, 6H), 2.07 (m, 1H), 2.58 (m, 1H), 3.02 (m, 1H),3.20-3.80 (m, 24H), 6.99 (s, 1H), 7.02 (d, 1H), 7.12 (d, 1H), 7.28 (s,1H), 7.64 (d, 1H), 8.86 (d, 1H)

Example 97(E)-3-{2-(4-[2-(4-Aza-1-azoniabicyclo[2.2.2]oct-1-yl)acetyl]piperazino-8-(2-[4-(tert-butyl)-1,3-thiazol-2-yl]ethyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

¹H-NMR (CD₃OD): 1.29 (s, 9H), 3.20-3.41 (m, 5H), 3.50-3.79 (m, 20H),4.39 (s, 2H), 6.96 (s, 1H), 7.02 (d, 1H), 7.13 (d, 1H), 7.29 (s, 1H),7.64 (d, 1H), 8.86 (d, 1H)

MS (ES+): 620

Example 98N-((E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoyl)methanesulfonamide

(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoicacid (24 mg) was dissolved in dimethylformamide (2 ml), added withmethanesulfonamide (15 mg), dimethylaminopyridine (20 mg) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (31 mg), andthen the mixture was stirred at room temperature for 24 hours. Thereaction solution was added with ethyl acetate and hexane, washed with0.2 M hydrochloric acid, and dried over sodium over sodium sulfate, andthe solvent was evaporated under reduced pressure. The residue waspurified by silica gel column chromatography to obtain the titlecompound (14 mg) as yellow powder.

¹H-NMR (CDCl₃) δ: 1.21 (d, 6H), 3.02 (m, 1H), 3.15 (m, 2H), 3.19 (s,3H), 3.36 (m, 2H), 3.53 (m, 4H), 3.70 (m, 4H), 6.78 (s, 1H), 6.86 (d,1H), 6.92 (d, J=16Hz, 1H), 7.17 (s, 1H), 7.53 (d, J=16Hz, 1H), 8.67 (d,1H)

MS (ES+) m/z 532 (M⁺+1)

Example 99N-((E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-2-propenoyl)methanesulfonamide

¹H-NMR (CDCl₃) δ: 1.28 (d, 6H), 3.04 (m, 1H), 3.11 (s, 3H), 3.37 (m,4H), 6.73 (s, 1H), 7.19 (d, J=7Hz, 1H), 7.32 (d, J=14Hz, 1H), 7.59 (s,1H), 7.72 (d, J=14Hz, 1H), 8.48 (s, 1H), 9.08 (d, J=7Hz, 1H)

MS (ES+) m/z 447 (M⁺+1); MS (ES−) m/z 445 (M⁺−1)

Example 100N-((E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoyl)-3-amino-1-propanesulfonamide

(A)N-((E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoyl)-3-(tert-butoxycarbonylamino)-1-propanesulfonamide

(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoicacid (53 mg) was dissolved in dimethylformamide (2 ml), added with3-(N-tert-butoxycarbonylamino)propanesulfonamide (55 mg) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (67 mg), andthe mixture was stirred at room temperature for 24 hours. The reactionsolution was added with ethyl acetate and hexane, washed with 0.2 Mhydrochloric acid, and dried over sodium sulfate, and the solvent wasevaporated under reduced pressure. The residue was purified by silicagel column chromatography to obtain the title compound (32 mg) as yellowpowder.

¹H-NMR (300MHz) δ: 1.22 (d, J=7.2Hz, 6H), 1.40 (s, 9H), 1.90-2.05 (m,2H), 3.00-3.80 (m, 17H), 6.70 (s, 1H), 6.85 (d, J=7.5Hz, 1H), 7.20 (s,1H), 7.35 (d, J=15.4Hz, 1H), 7.65 (d, J=15.4Hz, 1H), 9.00-9.10 (m, 2H)

(B)N-((E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido-[1,2-a]pyrimidin-3-yl}-2-propenoyl)-3-amino-1-propanesulfonamide

TheN-((E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoyl)-3-(tert-butoxycarbonylamino)-1-propanesulfonamide(32 mg) obtained in (A) was dissolved in trifluoroacetic acid (2 ml) andstirred for 40 minutes. The solvent was evaporated under reducedpressure, and the residue was purified by medium pressure reverse phasecolumn chromatography to obtain the title compound (quantitative).

¹H-NMR (CD₃OD) δ: 1.25 (d, 6H), 2.18 (m, 2H), 3.02 (m, 1H), 3.1-3.45 (m,with CHD₂OD), 3.60 (m, 4H), 3.80 (m, 4H), 6.98 (s, 1H), 7.12 (s, 1H),7.16 (d, J=16Hz, 1H), 7.26 (s, 1H), 7.75 (d, J=16Hz, 1H), 8.82 (d, 1H)

MS (ES+) m/z 575 (M⁺+1); MS (ES−) m/z 573 (M⁺−1)

Example 101(E)-3-{8-[(E)-2-(4-Isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-{[(4-methylphenyl)sulfonyl]oxy}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

4-[(E)-2-(4-Isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-pyridinamine (100mg, 0.408 mmol) was dissolved in xylene (1.2 ml), added withtrichlorophenyl malonate (208 mg, 0.448 mmol), and then the mixture wasrefluxed by heating for 1.3 hours. The reaction mixture was added withn-hexane, and the deposited solid was collected by filtration.Dimethylformamide (110 μl) was added with phosphorus oxychloride (190μl, 2.04 mmol) under ice cooling and stirred at room temperature for 30minutes. The mixture was added with the solid dissolved indichloromethane (3.0 ml) under ice cooling, and stirred at roomtemperature for 1.5 hours. The reaction mixture was added with saturatedaqueous sodium hydrogencarbonate and extracted with chloroform. Theorganic layer was dried over magnesium sulfate, and the solvent wasevaporated under reduced pressure. The residue was suspended inanhydrous tetrahydrofuran (4.0 ml), added with(tert-butoxycarbonylmethylene)triphenylphosphorane (460 mg, 1.22 mmol),and stirred at 80° C. for 5 days. The reaction solution wasconcentrated, and then the residue was purified by silica gel columnchromatography (chloroform:methanol=100:0→100:10, v/v) and thin layerchromatography (chloroform:methanol=10:1, v/v). The resulting compoundwas dissolved in anhydrous tetrahydrofuran (600 μl) anddimethylformamide (600 μl), added with 4-dimethylaminopyridine (10.2 mg,0.0831 mmol) and p-toluenesulfonyl chloride (13.4 mg, 0.0703 mmol), andstirred at room temperature for 2 hours. The reaction mixture was addedwith water and extracted with chloroform. The organic layer was driedover magnesium sulfate, and the solvent was evaporated under reducedpressure. The residue was purified by thin layer chromatography(n-hexane:ethyl acetate=2:1, v/v) to obtain the title compound (9.0 mg,3.7%).

¹H-NMR (CDCl₃) δ: 1.37 (6H, d, J=6.8Hz), 1.51 (9H, s), 2.48 (3H, s),3.14-3.21 (1H, m), 7.03 (1H, s), 7.18 (1H, d, J=15.9Hz), 7.38-7.42 (4H,m), 7.52 (1H, d, J=1.5Hz), 7.56 (1H, d, J=16.1Hz), 7.67 (1H, d,J=15.9Hz), 8.05 (2H, d, J=8.3Hz), 9.02 (1H, d, J=7.6Hz)

(B) tert-Butyl(E)-3-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

The tert-butyl(E)-3-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-{[(4-methylphenyl)sulfonyl]oxy}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(9.0 mg, 0.0152 mmol) obtained in (A) was dissolved in dimethylformamide(0.75 ml), added with morpholine (13.2 μl, 0.152 mmol), and stirredovernight at room temperature. The reaction mixture was concentrated,and then the residue was purified by thin layer chromatography(n-hexane:ethyl acetate=1:1, v/v) to obtain the title compound (6.7 mg,87%).

¹H-NMR (CDCl₃) δ: 1.33 (6H, d, J=6.8Hz), 1.52 (9H, s), 3.12-3.19 (1H,m), 3.62-3.64 (4H, m), 3.73-3.75 (4H, m), 6.97 (1H, s), 7.08 (1H, d,J=15.9Hz), 7.16 (1H, dd, J=1.5, 7.6Hz), 7.34-7.38 (2H, m), 7.50 (2H, d,J=15.9Hz), 8.91 (1H, d, J=7.6Hz)

(C)(E)-3-{8-[(E)-2-(4-Isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-butyl(E)-3-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(6.7 mg, 0.0132 mmol) obtained in (B) was dissolved in 1,4-dioxane (260μl), added with 4 N hydrochloric acid solution in 1,4-dioxane (260 μl),and stirred at room temperature for 7 hours. The reaction solution wasconcentrated, and the residue was purified by thin layer chromatography(chloroform:methanol=10:1, v/v) to obtain the title compound (4.8 mg,81%).

¹H-NMR (CDCl₃+CD₃OD) δ: 1.36 (6H, d, J=6.8Hz), 3.13-3.20 (1H, m), 3.67(4H, t, J=4.4Hz), 3.86 (4H, t, J=4.4Hz), 7.04 (1H, d, J=15.6Hz), 7.07(1H, s), 7.33 (1H, dd, J=1.6, 7.5Hz), 7.40 (1H, d, J=16.2Hz), 7.47 (1H,d, J=6.6Hz), 7.58 (1H, d, J=16.2Hz), 7.62 (1H, d, J=15.6Hz), 8.89 (1H,d, J=7.5Hz)

FAB-MS; m/z: 453 (M⁺+1)

FAB-HRMS; Calcd. for C₂₃H₂₅O₄N₄S+H+: 453.1597, Found: 453.1602

Example 102(E)-3-{2-(3-Hydroxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) 4-Isopropyl-1,3-thiazole-2-carbaldehyde

(4-Isopropyl-1,3-thiazol-2-yl)methanol (5.20 g, 33.0 mmol) was dissolvedin methylene chloride (100 ml), added with pyridinium dichromate (14.9g, 39.7 mmol), and stirred at room temperature for 19 hours stirred.After insoluble solids were removed, the reaction solution wasevaporated, and the residue was purified by silica gel columnchromatography (chloroform) to obtain the title compound (3.32 g, 65%).

¹H-NMR (CDCl₃) δ: 1.37 (6H, d, J=6.8Hz), 3.18-3.25 (1H, m), 7.34 (1H,s), 9.98 (1H, s)

(B) tert-ButylN-4-[2-hydroxy-2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-pyridylcarbamate

tert-Butyl N-(4-methyl-2-pyridyl)carbamate (6.24 g, 30.0 mmol) wasdissolved in anhydrous tetrahydrofuran (100 ml), added with n-butyllithium (1.59 M in n-hexane, 47.1 ml, 74.9 mmol) at −78° C. under argonatmosphere, and then stirred at room temperature for 1.5 hours. Thereaction solution was cooled to −78° C. again, then added with asolution of the 4-isopropyl-1,3-thiazole-2-carbaldehyde (4.65 g, 30.0mmol) obtained in (A) in anhydrous tetrahydrofuran (50.0 ml), and thenthe mixture was stirred for 3 hours. The reaction mixture was added withsaturated aqueous ammonium chloride, warmed to room temperature, andthen extracted with ethyl acetate. The organic layer was dried overmagnesium sulfate, and the solvent was evaporated under reducedpressure. The residue was purified by silica gel column chromatography(n-hexane:ethyl acetate=3:1→1:1, v/v) to obtain the title compound (5.42g, 50%).

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.8Hz), 1.53 (9H, s), 3.03-3.11 (2H,m), 3.28-3.32 (2H, m), 5.24 (1H, dd, J=4.2, 8.5Hz), 6.82 (1H, d,J=0.7Hz), 6.85 (1H, dd, J=1.6, 5.2Hz), 7.90 (1H, s), 8.10 (1H, s), 8.14(1H, d, J=5.2Hz)

ESI-MS; m/z: 364 (M⁺+1)

(C) tert-ButylN-4-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-pyridylcarbamate

The tert-butylN-4-[2-hydroxy-2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-pyridylcarbamate(5.30 g, 14.6 mmol) obtained in (B) was dissolved in anhydroustetrahydrofuran (100 ml), added with triethylamine (5.08 ml, 36.5 mmol)and methanesulfonyl chloride (1.35 ml, 17.5 mmol), and then the mixturewas stirred at room temperature for 1 hour. After insoluble solids wereremoved, the reaction solution was washed with tetrahydrofuran, and thesolvent was evaporated under reduced pressure. The residue was dissolvedin toluene (100 ml), added with 1,8-diazabicyclo[5.4.0]undec-7-ene (10.9ml, 72.9 mmol), and refluxed by heating for 30 minutes. The reactionsolution was concentrated, then added with 1 N hydrochloric acid andextracted with chloroform. The organic layer was washed with saturatedaqueous sodium hydrogencarbonate and dried over magnesium sulfate, andthe solvent was evaporated under reduced pressure. The residue waspurified by silica gel column chromatography (n-hexane:ethylacetate=5:1→3:1, v/v) to obtain the title compound (3.76 g, 75%).

¹H-NMR (CDCl₃) δ: 1.34 (6H, d, J=6.8Hz), 1.56 (9H, s), 3.07-3.18 (1H,m), 6.87 (1H, s), 7.07 (1H, dd, J=1.2, 5.1Hz), 7.31 (1H, d, J=16.1Hz),7.50 (1H, d, J=16.1Hz), 8.12 (1H, s), 8.25 (1H, s), 8.26 (1H, s)

(D) 4-[(E)-2-(4-Isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-pyridinamine

The tert-butylN-4-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-pyridylcarbamate(3.75 g, 10.9 mmol) obtained in (C) was dissolved in dichloromethane(50.0 ml), added with trifluoroacetic acid (50.0 ml), and then themixture was stirred at room temperature for 30 minutes. The reactionsolution was concentrated, then neutralized with saturated aqueoussodium hydrogencarbonate, and extracted with chloroform. The organiclayer was dried over magnesium sulfate, and then the solvent wasevaporated under reduced pressure to obtain the title compound (2.65 g,100%).

¹H-NMR (CDCl₃) δ: 1.34 (6H, d, J=6.8Hz), 3.09-3.16 (1H, m), 4.67 (1H, brs), 6.57 (1H, s), 6.80 (1H, dd, J=1.3, 5.5Hz), 6.87 (1H, s), 7.18 (1H,d, J=16.1Hz), 7.37 (1H, d, J=16.1Hz), 8.04 (1H, d, J=5.5Hz)

(E)2-Hydroxy-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-4-one

The 4-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-pyridinamine(2.65 g, 10.8 mmol) obtained in (D) was dissolved in toluene (100 ml),added with trichlorophenyl malonate (5.50 g, 11.9 mmol), and refluxed byheating for 1.5 hours. After the reaction mixture was concentrated, thedeposited solid was collected by filtration and washed with n-hexane andether to obtain the title compound (3.14 g, 93%) as yellow solid.

¹H-NMR (CDCl₃) δ: 1.37 (6H, d, J=6.8Hz), 3.14-3.21 (1H, m), 5.41 (1H,s), 7.05 (1H, s), 7.40 (1H, dd, J=1.5, 7.4Hz), 7.46 (1H, d, J=16.1Hz),7.51 (1H, d, J=1.5Hz), 7.66 (1H, d, J=16.1Hz), 9.09 (1H, d, J=7.4Hz)

(F)2-(3-Hydroxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-4-one

The2-hydroxy-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-4-one(1.00 g, 3.19 mmol) obtained in (E) was dissolved in anhydroustetrahydrofuran (15.0 ml) and anhydrous dimethylformamide (15.0 ml),added with 4-dimethylaminopyridine (585 mg, 4.79 mmol) andp-toluenesulfonyl chloride (730 mg, 3.83 mmol), and then the mixture wasstirred at room temperature for 50 minutes. Subsequently, the reactionsolution was added with 3-hydroxypiperidine (646 mg, 6.38 mmol) andstirred at room temperature for 16 hours, and then added with3-hydroxypiperidine (968 mg, 9.57 mmol) and stirred at 60° C. for 1.5hours. After the reaction mixture was concentrated, the residue waspurified by silica gel column chromatography(chloroform:methanol=100:0→100:1→100:2→100:5→100:10, v/v) to obtain thetitle compound (865 mg, 68%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=7.1Hz), 1.54-1.72 (1H, m), 1.86-2.01(3H, m), 3.11-3.21 (1H, m), 3.39-3.45 (1H, m), 3.55 (1H, dd,J=7.1,13.2Hz), 3.73-3.78 (1H, m), 3.85-3.89 (1H, m), 4.00 (1H, dd,J=3.1,12.9Hz), 5.65 (1H, m), 6.94 (1H, m), 7.03 (1H, dd, J=2.0, 7.6Hz),7.32 (1H, d, J=16.1Hz), 7.45 (1H, d, J=16.1Hz), 8.82 (1H, d, J=7.3Hz)

ESI-MS; m/z: 397 (M⁺+1)

(G) tert-Butyl(E)-3-{2-(3-formyloxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

Dimethylformamide (15.0 ml) was added with phosphorus oxychloride (608μl, 6.52 mmol) under ice cooling and stirred at room temperature for 40minutes. The mixture was added to a solution of the2-(3-hydroxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-4-one(862 mg, 2.17 mmol) obtained in (F) in dimethylformamide (15.0 ml) underice cooling, and then the mixture was stirred at room temperature for1.5 hours. The reaction mixture was added with saturated aqueous sodiumhydrogencarbonate and extracted with chloroform. The organic layer wasdried over magnesium sulfate, and the solvent was evaporated underreduced pressure. The residue was suspended in anhydrous tetrahydrofuran(20 ml) and anhydrous N,N-dimethylformamide (10 ml), added with(tert-butoxycarbonylmethylene)triphenylphosphorane (1.64 g, 4.35 mmol),and stirred at 50° C. for 16 hours. The reaction mixture wasconcentrated, and the residue was purified by silica gel columnchromatography (n-hexane:ethyl acetate=10:1→5:1→3:1→2:1, v/v) to obtainthe title compound (488 mg, 41%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=6.8Hz), 1.52 (9H, s), 1.72-1.75 (1H,m), 1.81-1.85 (1H, m), 1.94-2.03 (2H, m), 3.12-3.18 (1H, m), 3.51-3.60(2H, m), 3.71 (1H, dd, J=6.3, 13.4Hz), 3.82 (1H, dd, J=3.1,13.4Hz),5.13-5.16 (1H, m), 6.97 (1H, s), 7.07 (1H, d, J=15.6Hz), 7.15 (1H, dd,J=1.7, 7.6Hz), 7.34-7.37 (2H, m), 7.49 (1H, d, J=16.1Hz), 7.51 (1H, d,J=15.6Hz), 8.10 (1H, s), 8.89 (1H, d, J=7.6Hz)

ESI-MS; m/z: 551 (M⁺+1)

(H) tert-Butyl(E)-3-{2-(3-hydroxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

The tert-butyl(E)-3-{2-(3-formyloxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(184 mg, 0.334 mmol) obtained in (G) was dissolved in methanol (3.0 ml),added with 1 N aqueous sodium hydroxide (1.0 ml), and then the mixturewas stirred at room temperature for 10 minutes. The reaction mixture wasadded with 1 N hydrochloric acid (1.0 ml) and extracted with chloroform.The organic layer was dried over magnesium sulfate and the solvent wasevaporated under reduced pressure. The residue was purified by silicagel column chromatography (n-hexane:ethyl acetate=2:1→2:1→1:2, v/v) toobtain the title compound (174 mg, 100%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=6.8Hz), 1.52 (9H, s), 1.68-1.89 (4H,m), 3.12-3.18 (1H, m), 3.55-3.66 (3H, m), 3.93-3.96 (1H, m), 4.03 (1H,br s), 6.97 (1H, s), 7.06 (1H, d, J=15.6Hz), 7.16 (1H, dd, J=1.6,7.5Hz), 7.34 (1H, d, J=16.1Hz), 7.34 (1H, d, J=1.6Hz), 7.49 (1H, d,J=16.1Hz), 7.50 (1H, d, J=15.6Hz), 8.89 (1H, d, J=7.5Hz)

ESI-MS; m/z: 523 (M⁺+1)

(I)(E)-3-{2-(3-Hydroxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-Butyl(E)-3-{2-(3-hydroxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(43.4 mg, 0.0830 mmol) obtained in (H) was dissolved in 1,4-dioxane (200μl ), added with 4 N hydrochloric acid solution in 1,4-dioxane, andstirred at room temperature for 3.5 hours. The reaction solution wasconcentrated, and the residue was purified by thin layer chromatography(chloroform:methanol:water=10:1:0→8:3:0.1→7:3:1, v/v) to obtain thetitle compound (18.9 mg, 49%).

¹H-NMR (DMSO-d₆) δ: 1.29 (6H, d, J=6.6Hz), 1.42-1.47 (1H, m), 1.55-1.59(1H, m), 1.81 (1H, m), 1.90-1.95 (1H, m), 2.97-3.74 (4H, m), 3.89-3.92(1H, m), 4.95 (1H, br s), 6.89 (1H, d, J=15.1Hz), 7.42-7.46 (2H, m),7.54-7.62 (3H, m), 7.87 (1H, d, J=16.6Hz), 8.76 (1H, d, 7.8Hz)

ESI-MS; m/z: 467 (M⁺+1)

Example 103(E)-3-(2-{3-[(Aminocarbonyl)oxy]piperidino}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A) tert-Butyl(E)-3-(2-{3-[(aminocarbonyl)oxy]piperidino}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

tert-Butyl(E)-3-{2-(3-hydroxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(138 mg, 0.264 mmol) was dissolved in ethyl acetate (2.6 ml), added withtrichloroacetyl isocyanate (62.6 μl, 0.528 mmol) under ice cooling, andthen the mixture was stirred at room temperature for 15 minutes. Thereaction mixture was concentrated, dissolved in methanol (5.0 ml) andwater (0.5 ml), and then added with sodium formate (71.8 mg, 1.06 mmol),and stirred overnight at room temperature. The reaction mixture wasfurther added with chloroform (3.0 ml) and sodium formate (71.8 mg, 1.06mmol) and stirred overnight. The reaction mixture was concentrated, andthe residue was purified by silica gel column chromatography(chloroform:methanol=100:0 100:1 100:2, v/v) to obtain the titlecompound (150 mg, 100%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=6.8Hz), 1.50 (9H, s), 1.70-2.04 (4H,m), 3.11-3.18 (1H, m), 3.27-3.33 (1H, m), 3.52-3.56 (1H, m), 3.68-3.74(2H, m), 4.85 (1H, br s), 5.15 (2H, br s), 6.97 (1H, s), 7.11 (1H, d,J=15.6Hz), 7.15 (1H, dd, J=1.8, 7.6Hz), 7.34 (1H, d, J=16.5Hz), 7.37(1H, s), 7.49 (1H, d, J=16.5Hz), 7.72 (1H, d, J=15.6Hz), 8.89 (1H, d,J=7.6Hz)

ESI-MS; m/z: 566 (M⁺+1)

(B)(E)-3-(2-{3-[(Aminocarbonyl)oxy]piperidino}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

The tert-butyl(E)-3-(2-{3-[(aminocarbonyl)oxy]piperidino}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoateobtained in (B) was dissolved in 1,4-dioxane (100 μl), added with 4 Nhydrochloric acid solution in 1,4-dioxane (400 μl), and stirred at roomtemperature for 15 hours. The reaction solution was further added with 4N hydrochloric acid solution in 1,4-dioxane (400 μl) and stirred at roomtemperature for 7 hours. The reaction solution was concentrated, and theresidue was subjected to azeotropy with toluene. The product was addedwith saturated aqueous sodium hydrogencarbonate, neutralized withphosphate buffer (pH 7-8), and extracted with a mixture ofchloroform/methanol (10:1, v/v). The organic layer was dried overmagnesium sulfate, and the solvent was evaporated under reducedpressure. The residue was purified by thin layer chromatography(chloroform:methanol=20:1, v/v) to obtain the title compound (8.1 mg,36%).

¹H-NMR (CDCl₃+CD₃OD) δ: 1.35 (6H, d, J=7.1Hz), 1.71 (1H, m), 1.90-1.96(3H, m), 3.12-3.19 (1H, m), 3.38-3.40 (1H, m), 3.58-3.81 (3H, m), 4.87(1H, br s), 6.99 (1H, s), 7.11 (1H, d, J=15.6Hz), 7.21 (1H, d, J=7.7Hz),7.35 (1H, d, J=16.1Hz), 7.40 (1H, s), 7.52 (1H, d, J=16.1Hz), 7.76 (1H,d, J=15.6Hz), 8.88 (1H, d, J=7.7Hz)

ESI-MS; m/z: 510 (M⁺+1)

Example 1042-(3-Hydroxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-4-one

(A) tert-Butyl(E)-3-{2-[(3-acetyloxy)piperidino]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl(E)-3-{2-(3-formyloxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-.3-yl}-2-propenoate(115 mg, 0.209 mmol) was dissolved in methanol (2.0 ml), added with 1 Naqueous sodium hydroxide (627 μl), and stirred at room temperature for10 minutes. The reaction solution was added with 1 N hydrochloric acid(627 μl) and extracted with chloroform. The organic layer was dried overmagnesium sulfate, and the solvent was evaporated under reducedpressure. The residue was dissolved in dichloromethane (2.0 ml), addedwith 4-dimethylaminopyridine (51.0 mg, 0.418 mmol) and acetic anhydride(29.6 μl), and then the mixture was stirred at room temperature for 10minutes. The reaction mixture was added with 1 N hydrochloric acid andextracted with chloroform. The organic layer was dried over magnesiumsulfate, and the solvent was evaporated under reduced pressure. Theresidue was purified by silica gel column chromatography (n-hexane:ethylacetate=5:1→3:1→2:1, v/v) to obtain the title compound (121 mg,quantitative).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=6.8Hz), 1.52 (9H, s), 1.70-1.78 (2H,m), 1.90-2.02 (2H, m), 2.06 (3H, s), 3.12-3.19 (1H, m), 3.47-3.52 (1H,m), 3.61 (2H, dd, J=7.1, 13.2Hz), 3.88 (1H, dd, J=3.3, 13.2Hz),4.96-5.00 (1H, m), 6.96 (1H, s), 7.06 (1H, d, J=15.6Hz), 7.13 (1H, dd,J=2.0, 7.6Hz), 7.33-7.37 (2H, m), 7.48 (1H, d, J=16.1Hz), 7.50 (1H, d,J=15.6Hz), 7.50 (1H, d, J=15.6Hz), 8.89 (1H, d, J=7.6Hz)

ESI-MS; m/z: 565 (M⁺+1)

(B)1-(3-{(E)-3-[(2-Cyanoethyl)amino]-3-oxo-1-propenyl}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-3-piperidylacetate

The tert-Butyl(E)-3-{2-[(3-acetyloxy)piperidino]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(121 mg, 0.214 mmol) obtained in (A) was dissolved in 1,4-dioxane (1.0ml), added with 4 N hydrochloric acid solution in 1,4-dioxane, and thenthe mixture was stirred overnight at room temperature. The reactionmixture was concentrated, and the residue was suspended indichloromethane (2.0 ml), added with 2-cyanoethylamine (79 μl, 1.07mmol), BOPCl (109 mg, 0.429 mmol) and diisopropylethylamine (187 μl,1.07 mmol), and stirred for 1 hour. The reaction solution was furtheradded with 2-cyanoethylamine (79 μl, 1.07 mmol), BOPCl (109 mg, 0.429mmol) and diisopropylethylamine (187 μl, 1.07 mmol) and stirredovernight. The reaction mixture was added with saturated aqueous sodiumhydrogencarbonate and extracted with a mixture of chloroform/methanol(10:1, v/v). The organic layer was dried over magnesium sulfate, and thesolvent was evaporated under reduced pressure. The residue was purifiedby column chromatography (chloroform:methanol, 100:0→100:1→100:2, v/v)to obtain the title compound (53.3 mg, 48%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=6.8Hz), 1.70-1.79 (2H, m), 1.90-1.93(1H, m), 2.01-2.04 (1H, m), 2.06 (3H, s), 2.71 (2H, t, J=6.5Hz),3.11-3.18 (1H, m), 3.46-3.51 (1H, m), 3.56-3.68 (4H, m), 3.92 (1H, dd,J=3.1,13.1Hz), 4.95-5.00 (1H, m), 6.58-6.61 (1H, m), 6.96 (1H, s), 7.13(1H, d, J=7.6Hz), 7.25 (1H, d, J=15.1Hz), 7.31-7.34 (2H, m), 7.47 (1H,d, J=16.4Hz), 7.47 (1H, d, J=15.1Hz), 7.52 (1H, d, J=15.1Hz), 8.83 (1H,d, J=7.6Hz)

ESI-MS; m/z: 561 (M⁺+1)

(C)1-(3-{(E)-2-[1-(2-Cyanoethyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-3-piperidylacetate

The1-(3-{(E)-3-[(2-cyanoethyl)amino]-3-oxo-1-propenyl}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-3-piperidylacetate (57.3 mg, 0.102 mmol) obtained in (B) was suspended inacetonitrile (3.0 ml), added with sodium azide (13.3 mg, 0.204 mmol) andtrifluoromethanesulfonic acid anhydride (25.8 μl, 0.153 mmol) under icecooling, and then the mixture was stirred at room temperature for 1hour. The reaction mixture was further added with sodium azide (13.3 mg,0.204 mmol) and trifluoromethanesulfonic acid anhydride (25.8 μl, 0.153mmol), and stirred for 3 hours. The reaction mixture was added withsaturated aqueous sodium hydrogencarbonate and extracted withchloroform, and the organic layer was dried over magnesium sulfate. Thesolvent was evaporated under reduced pressure, and the residue waspurified by thin layer chromatography (hexane:ethyl acetate=1:2→1:3→0:1,v/v) to obtain the title compound (26.8 mg, 45%).

¹H-NMR (CDCl₃) δ: 1.36 (6H, d, J=6.9Hz), 1.73-1.80 (4H, m), 2.04 (3H,s), 3.10 (2H, t, J=7.0Hz), 3.13-3.19 (1H, m), 3.57-3.73 (3H, m), 3.91(1H, dd, J=2.8, 13.1Hz), 4.69 (2H, t, J=7.0Hz), 4.95-4.98 (1H, m), 6.97(1H, s), 7.17 (1H, dd, J=1.5, 7.5Hz), 7.36 (1H, d, J=1.5Hz), 7.36 (1H,d, J=16.0Hz), 7.50 (1H, d, J=16.0Hz), 7.75 (1H, d, J=15.3Hz), 7.83 (1H,d, J=15.3Hz), 8.86 (1H, d, J=7.5Hz)

(D)2-(3-Hydroxypiperidino)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-3-[(E)-2-(1H-1,2,3,4-tetraazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-4-one

The1-(3-{(E)-2-[1-(2-cyanoethyl)-1H-1,2,3,4-tetraazol-5-yl]-1-ethenyl}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-3-piperidylacetate (25.9 mg, 0.0442 mmol) obtained in (C) was suspended in methanol(0.5 ml) and anhydrous tetrahydrofuran (1.0 ml), added with sodiummethoxide (4.8 mg, 0.0884 mmol) under ice cooling, and then stirred for4 hours. The reaction solution was further added with sodium methoxide(4.8 mg, 0.0884 mmol) and stirred for 1 hour. The reaction mixture wasadded with saturated aqueous ammonium chloride and extracted withchloroform, and the organic layer was dried over magnesium sulfate.Then, the solvent was evaporated under reduced pressure, and the residuewas purified by thin layer chromatography (chloroform:methanol=10:1,v/v) to obtain the title compound (12.7 mg, 59%).

¹H-NMR (CD₃OD) δ: 1.32 (6H, d, J=7.1Hz), 1.56-1.61 (1H, m), 1.67-1.71(1H, m), 1.89-1.92 (1H, m), 2.03-2.07 (1H, m), 3.08-3.19 (2H, m), 3.45(1H, s), 3.76-3.80 (1H, m), 3.84-3.88 (1H, m), 4.00 (1H, dd, J=2.9,12.9Hz), 7.19 (1H, s), 7.34-7.36 (2H, m), 7.39 (1H, d, J=16.4Hz), 7.51(1H, d, J=16.1Hz), 7.57 (1H, d, J=16.4Hz), 7.74 (1H, d, J=16.1Hz), 8.76(1H, d, J=7.6Hz)

ESI-MS; m/z: 491 (M⁺+1)

Example 105(E)-3-{2-[(3R)-3-Hydroxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-{2-[(3R)-3-Formyloxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

2-Hydroxy-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-4-one(500 mg, 1.60 mmol) was treated in the same manner as in Example 102,(F) and (G) to obtain the title compound (488 mg, 56%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=6.8Hz), 1.52 (9H, s), 1.73-1.75 (1H,m), 1.83-1.85 (1H, m), 1.95-2.03 (2H, m), 3.13-3.17 (1H, m), 3.54-3.60(2H, m), 3.71 (1H, dd, J=6.2, 13.5Hz), 3.82 (1H, dd, J=3.2, 13.5Hz),5.14-5.16 (1H, m), 6.97 (1H, s), 7.07 (1H, d, J=15.6Hz), 7.14 (1H, dd,J=1.6, 7.6Hz), 7.34-7.37 (2H, m), 7.49 (1H, d, J=16.0Hz), 7.51 (1H, d,J=15.6Hz), 8.10 (1H, s), 8.89 (1H, d, J=7.6Hz)

(B) tert-Butyl(E)-3-{2-[(3R)-3-Hydroxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

The tert-butyl(E)-3-{2-[(3R)-3-formyloxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(488 mg, 0.886 mmol) obtained in (A) was treated in the same manner asin Example 102, (H) to obtain the title compound (373 mg, 81%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=6.8Hz), 1.52 (9H, s), 1.82-1.88 (4H,m), 3.11-3.18 (1H, m), 3.54-3.67 (3H, m), 3.95-3.98 (1H, m), 4.03 (1H,br s), 6.97 (1H, s), 7.05 (1H, d, J=15.7Hz), 7.16 (1H, dd, J=1.8,7.6Hz), 7.34 (1H, d, J=16.1Hz), 7.34 (1H, s), 7.49 (1H, d, J=16.1Hz),7.50 (1H, d, J=15.7Hz), 8.89 (1H, d, J=7.6Hz)

(C)(E)-3-{2-[(3R)-3-Hydroxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

tert-Butyl(E)-3-{2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(105 mg, 0.201 mmol) obtained in (B) was treated in the same manner asin Example 102, (I) to obtain the title compound (64.0 mg, 68%).

¹H-NMR (DMSO-d₆) δ: 1.27 (6H, d, J=6.8Hz), 1.40-1.45 (1H, m), 1.54-1.57(1H, m), 1.80-1.83 (1H, m), 1.89-1.93 (1H, m), 2.96-3.19 (2H, m), 3.60(1H, m), 3.88-3.90 (1H, m), 4.92 (1H, br s), 6.87 (1H, d, J=15.5Hz),7.40 (1H,S), 7.43 (1H, d, J=15.5Hz), 7.54 (1H, d, J=16.1Hz), 7.58-7.59(2H, m), 7.85 (1H, d, J=16.1Hz), 8.74 (1H, d, 8.1Hz)

Example 106(E)-3-{2-[(3S)-3-Hydroxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-{2-[(3S)-3-formyloxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

2-Hydroxy-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-4-one(500mg, 1.60 mmol) was treated in the same manner as in Example 102, (F) and(G) to obtain the title compound (164 mg, 19%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=6.9Hz), 1.52 (9H, s), 1.73-1.75 (1H,m), 1.81-1.85 (1H, m), 1.96-2.05 (2H, m), 3.11-3.18 (1H, m), 3.54-3.57(2H, m), 3.70 (1H, dd, J=6.5, 13.1Hz), 3.82 (1H, d, J=13.5Hz), 5.15 (1H,m), 6.96 (1H, s), 7.06 (1H, d, J=15.4Hz), 7.14 (1H, dd, J=1.6, 7.4Hz),7.32-7.36 (2H, m), 7.48 (1H, d, J=15.9Hz), 7.51 (1H, d, J=15.4Hz), 8.10(1H, s), 8.87 (1H, d, J=7.4Hz)

(B) tert-Butyl(E)-3-{2-[(3S)-3-hydroxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

The tert-butyl(E)-3-{2-[(3S)-3-formyloxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(164 mg, 0.298 mmol) obtained in (A) was treated in the same manner asin Example 102, (H) to obtain the title compound (156 mg, 100%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=6.8Hz), 1.52 (9H, s), 1.81-1.91 (4H,m), 3.11-3.18 (1H, m), 3.60-3.63 (3H, m), 3.86-3.89 (1H, m), 4.02 (1H,brs), 6.97 (1H, s), 7.04 (1H, d, J=15.6Hz), 7.13 (1H, dd, J=1.7, 7.6Hz),7.31 (1H, d, J=1.7Hz), 7.32 (1H, d, J=16.0Hz), 7.48 (1H, d, J=16.0Hz),7.49 (1H, d, J=15.6Hz), 8.87 (1H, d, J=7.6Hz)

(C)(E)-3-{2-[(3S)-3-Hydroxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-butyl(E)-3-{2-[(3S)-3-hydroxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(72.7 mg, 0.139 mmol) obtained in (B) was treated in the same manner asin Example 102, (I) to obtain the title compound (35.5 mg, 55%).

¹H-NMR (DMSO-d₆) δ: 1.29 (6H, d, J=6.8Hz), 1.42-1.45 (1H, m), 1.56-1.59(1H, m), 1.81-1.85 (1H, m), 1.93-1.95 (1H, m), 2.97-3.20 (3H, m), 3.62(1H, m), 3.69-3.74 (1H, m), 3.89-3.92 (1H, m), 4.90 (1H, br s), 6.89(1H, d, J=15.4Hz), 7.42-7.46 (1H, m), 7.55 (1H, d, J=16.2Hz), 7.61-7.62(2H, m), 7.87 (1H, d, J=16.2Hz), 8.76 (1H, d, 7.6Hz), 11.84 (1H, br s)

Example 107(E)-3-(2-{(3R)-3-[(Aminocarbonyl)oxy]hexahydro-1-pyridinyl}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-.4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

The tert-butyl(E)-3-(2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(109 mg, 0.209 mmol) was treated in the same manner as in Example 103,(A) and (B) to obtain the title compound (89.7 mg, 84%).

¹H-NMR (DMSO-d₆) δ: 1.29 (6H, d, J=6.9Hz), 1.66 (2H, m), 1.88-1.98 (2H,m), 3.05-3.12 (1H, m), 3.36-3.54 (3H, m), 3.86 (1H, d, J=10.0Hz), 4.60(1H, m), 6.47 (2H, br s), 6.90 (1H, d, J=15.2Hz), 7.42 (1H, s), 7.45(1H, d, J=15.2Hz), 7.55 (1H, d, J=16.2Hz), 7.62-7.64 (2H, m), 7.88 (1H,d, J=16.2Hz), 8.77 (1H, d, J=7.3Hz), 11.90 (1H, br s)

Example 108(E)-3-(2-{(3S)-3-[(Aminocarbonyl)oxy]hexahydro-1-pyridinyl)-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

The tert-butyl (E)-3-{2-[(3S)-3-hydroxyhexahydro-1pyridinyl]-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(83.6 mg, 0.160 mmol) was treated in the same manner as in Example 103(A) and (B) to obtain the title compound (59.4 mg, 73%).

¹H-NMR (DMSO-d₆) δ: 1.29 (6H, d, J=6.6Hz), 1.66 (2H, m), 1.88 (1H, m),1.98 (1H, m), 3.06-3.13 (1H, m), 3.43-3.51 (3H, m), 3.86 (1H, d,J=11.7Hz), 4.61 (1H, m), 6.47 (2H, brs), 6.90 (1H, d, J-15.6Hz), 7.42(1H, s), 7.46 (1H, d, J=15.6Hz), 7.55 (1H, d, J=16.1Hz), 7.62-7.64 (2H,m), 7.88 (1H, d, J=16.1Hz), 8.77 (1H, d, J=7.3Hz), 11.90 (1H, brs)

Example 109(E)-3-(2-(3-[(Dimethylamino)carbonyl]piperidino}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A) N³,N³-Dimethyl1-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-3-piperidinecarboxamide

The2-hydroxy-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4H-pyrido-[1,2-a]pyrimidin-4-one(164 mg, 0.523 mmol) obtained in Example 101, (E) was dissolved inanhydrous tetrahydrofuran (2.5 ml) and anhydrous dimethylformamide (2.5ml), added with 4-dimethylaminopyridine (83.0 mg, 0.680 mmol) andp-toluenesulfonyl chloride (110 mg, 0.576 mmol), and then the mixturewas stirred at 0° C. for 3 hours. Subsequently, the reaction solutionwas added with triethylamine (729 μl, 5.23 mmol) andN³,N³-dimethyl-3-piperidinecarboxamide trifluoroacetic acid salt (707mg, 2.62 mmol) and stirred at 0° C. for 1 hour, at room temperature for30 minutes and at ₆₀° C. for 19 hours. Then, the reaction solution wasadded with triethylamine (40.0 μl, 0.287 mmol) andN³,N³-dimethyl-3-piperidinecarboxamide trifluoroacetic acid salt (400mg, 1.48 mmol) and stirred at 60° C. for 5 hours. The reaction mixturewas concentrated, added with saturated aqueous ammonium chloride, andthen extracted with chloroform. The organic layer was dried over sodiumsulfate, and the solvent was evaporated under reduced pressure. Theresidue was purified by silica gel column chromatography(chloroform:methanol=100:1→100:3, v/v) to obtain the title compound (168mg, 71%).

¹H-NMR (CDCl₃) δ: 1.36 (6H, d, J=6.4Hz), 1.52-1.62 (1H, m), 1.73 (1H,s), 1.81-1.97 (3H, m), 2.70-2.76 (1H, m), 2.99 (3H, s), 3.14 (3H, s),3.04-3.18 (2H, m), 4.35 (1H, m), 4.65 (1H, m), 5.63 (1H, s), 6.95 (1H,s), 7.03 (1H, d, J=7.3Hz), 7.23 (1H, s), 7.32 (1H, d, J=16.1Hz), 7.45(1H, d, J=16.1Hz),-8.82 (1H, d, J=7.3Hz)

(B) tert-Butyl(E)-3-(2-{3-[(dimethylamino)carbonyl]piperidino}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

Dimethylformamide (3.0 ml) was added with phosphorus oxychloride (104μl, 1.12 mmol) under ice cooling and stirred at room temperature for 30minutes. This was added to theN³,N³-dimethyl-1-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}-3-piperidinecarboxamide(168 mg, 0.372 mmol) obtained in (A) and dissolved in dimethylformamide(4.0 ml) under ice cooling, and stirred at room temperature for 2 hours.The reaction mixture was concentrated, added with saturated aqueoussodium hydrogencarbonate, and then extracted with chloroform. Theorganic layer was dried over sodium sulfate, and the solvent wasevaporated under reduced pressure. The residue was suspended inanhydrous tetrahydrofuran (3 ml) and anhydrous N,N-dimethylformamide (3ml), added with (tert-butoxycarbonylmethylene)triphenylphosphorane (280mg, 0.744 mmol), and stirred at 50° C. for 37 hours. The reactionmixture was further added with(tert-butoxycarbonylmethylene)triphenylphosphorane (280 mg, 0.744 mmol),stirred at 80° C. for 5 hours, and then concentrated, and the residuewas purified by silica gel column chromatography(chloroform:methanol=100:1→100:2, v/v) to obtain the title compound (167mg, 78%).

¹H-NMR (CDCl₃) δ: 1.36 (6H, d, J=6.8Hz), 1.51 (9H, s), 1.65-1.68 (1H,m), 1.81-1.84 (1H, m), 1.89-1.94 (2H, m), 2.98 (3H, s), 3.04-3.17 (4H,m), 3.22 (3H, s), 4.05 (1H, m), 4.24 (1H, m), 6.96 (1H, s), 7.06 (1H, d,J=15.6Hz), 7.25 (1H, d, J=7.6), 7.34 (1H, d, J=16.1Hz), 7.46 (1H, s),7.47 (1H, d, J=16.1Hz), 7.52 (1H, d, J=15.6), 8.89 (1H, d, J=7.6Hz)

(C)(E)-3-(2-{3-[(Dimethylamino)carbonyl]piperidino}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

The tert-butyl(E)-3-(2-{3-[(dimethylamino)carbonyl]piperidino}-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate(71.0 mg, 0.123 mmol) obtained in (B) was added with trifluoroaceticacid (3.0 ml) and stirred at room temperature for 2 hours. Thetrifluoroacetic acid in the reaction mixture was evaporated underreduced pressure, and the residue was neutralized with 0.1 N sodiumhydroxide and phosphate buffer (pH 7-8) and extracted with chloroform.The organic layer was dried over sodium sulfate, and the solvent wasevaporated under reduced pressure. The residue was purified by thinlayer chromatography (chloroform:methanol and chloroform:methanol:water,8:3:1, v/v, lower layer) to obtain the title compound (39.0 mg, 61%).

¹H-NMR (DMSO-d₆) δ: 1.29 (6H, d, J=6.8Hz), 1.64 (1H, m), 1.72 (2H, m),1.87 (1H, m), 2.84 (3H, s), 3.04 (2H, m), 3.07-3.11 (2H, m), 3.15 (3H,s), 3.92 (1H, m), 4.08 (1H, m), 6.89 (1H, d, J=15.4), 7.42 (1H, s), 7.43(1H, d, J=15.4Hz), 7.56 (1H, d, J=16.1Hz), 7.60 (1H, s), 7.62 (1H, d,J=7.8Hz), 7.87 (1H, d, J=16.1Hz), 8.77 (1H, d, J=7.8Hz)

EI-MS; m/z: 522 (M⁺+1)

Example 110(E)-3-(8-[(E)-2-(4-Isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-{3-[(methylamino)carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A)N³-Methyl-1-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido-[1,2-a]pyrimidin-2-yl}-3-piperidinecarboxamide

2-Hydroxy-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-4-one(157 mg, 0.501 mmol) was dissolved in anhydrous tetrahydrofuran (2.5 ml)and anhydrous dimethylformamide (2.5 ml), added with4-dimethylaminopyridine (80.0 mg, 0.651 mmol) andp-toluenesulfonylchloride (105mg, 0.551 mmol), and stirred at 0° C. for1.5 hours. Subsequently, the reaction mixture was added withtriethylamine (698 μl, 5.01 mmol) and N³-methyl-3-piperidinecarboxamidehydrochloride (448 mg, 2.50 mmol), and then the mixture was stirred at60° C. for 5.5 hours. The reaction mixture was concentrated, added withsaturated aqueous ammonium chloride, and then extracted with chloroform.The organic layer was dried over sodium sulfate, and the solvent wasevaporated under reduced pressure. The residue was purified by silicagel column chromatography (chloroform:methanol=100:2→100:3, v/v) toobtain the title compound (174 mg, 79%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=6.8Hz), 1.60 (1H, m), 1.73 (1H, m),1.94 (1H, m), 2.02-2.05 (1H, m), 2.36-2.38 (1H, m), 2.83 (3H, d, J=4.9),3.13-3.20 (2H, m), 3.59 (1H, dd, J=8.8, 13.7), 4.00 (1H, m), 4.24 (1H,m), 5.62 (1H, s), 6.95 (1H, s), 7.05 (1H, d, J=7.8Hz), 7.27 (1H, s),7.33 (1H, d, J=16.1Hz), 7.46 (1H, d, J=16.1Hz), 8.83 (1H, d, J=7.8Hz)

(B) tert-Butyl(E)-3-(8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-{3-[(methylamino)carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

Dimethylformamide (3.0 ml) was added with phosphorus oxychloride (111μl, 1.19 mmol) under ice cooling and stirred at room temperature for 30minutes. The mixture was added to theN³-methyl-1-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}-3-piperidinecarboxamide(174 mg, 0.398 mmol) obtained in (A) and dissolved in dimethylformamide(4.0 ml) under ice cooling, and then the mixture was stirred at roomtemperature for 2 hours. The reaction mixture was concentrated, addedwith saturated aqueous sodium hydrogencarbonate, and then extracted withchloroform. The organic layer was dried over sodium sulfate, and thesolvent was evaporated under reduced pressure. The residue was suspendedin anhydrous tetrahydrofuran (3 ml) and dimethylformamide (3 ml), addedwith (tert-butoxycarbonylmethylene)triphenylphosphorane (449 mg, 1.19mmol) and stirred at 75° C. for 24 hours. The reaction mixture wasconcentrated, and the residue was purified by silica gel columnchromatography (chloroform:methanol=100:0→100:3, v/v) to obtain thetitle compound (192 mg, 86%).

¹H-NMR (CDCl₃) δ: 1.35 (6H, d, J=7.1Hz), 1.54 (9H, m), 1.65-1.68 (1H,m), 1.85-1.90 (1H, m), 1.95-2.00 (2H, m), 2.77 (3H, d, J=4.6), 2.82 (1H,m), 2.92 (1H, m), 3.11-3.17 (2H, m), 3.90-4.00 (1H, m), 4.45-4.50 (1H,m), 6.97 (1H, s), 7.09 (1H, d, J=15.6Hz), 7.12 (1H, d, J=1.7), 7.30 (1H,d, J=1.7Hz), 7.34 (1H, d, J=16.1Hz), 7.40-7.70 (2H, m), 8.86 (1H, d,J=7.3Hz)

(C)(E)-3-(8-[(E)-2-(4-Isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-(3-[(methylamino)-carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

The tert-butyl(E)-3-(8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-{3-[(methylamino)carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate (192 mg, 0.341 mmol) obtainedin (B) was added with trifluoroacetic acid (5.0 ml) and stirred at roomtemperature for 1.5 hours. After the reaction was completed, thetrifluoroacetic acid was evaporated under reduced pressure, and theresidue was neutralized with 0.1 N sodium hydroxide and phosphate buffer(pH 7-8) and extracted with chloroform. The organic layer was dried oversodium sulfate, and the solvent was evaporated under reduced pressure.The residue was purified by thin layer chromatography(chloroform:methanol and chloroform:methanol:water=8:3:1, v/v, lowerlayer) to obtain the title compound (49.0 mg, 28%).

¹H-NMR (DMSO-d₆) δ: 1.29 (6H, d, J=6.8Hz), 1.62-1.71 (2H, m), 1.77 (1H,m), 1.90 (1H, m), 2.58 (3H, d, J=4.6Hz), 3.05-3.13 (3H, m), 3.27 (1H,m), 3.89 (1H, m), 4.08 (1H, m), 6.89 (1H, d, J=15.6Hz), 7.42 (1H, s),7.42 (1H, d, J=15.6Hz), 7.54 (1H, d, J=16.1Hz), 7.62 (1H, d, J=7.6Hz),7.67 (1H, s), 7.79 (1H, s), 7.88 (1H, d, J=16.1Hz), 8.77 (1H, d,J=7.6Hz)

EI-MS; m/z: 508 (M⁺+1)

Example 1118-(E)-2-[4-(tert-Butyl)-1,3-thiazol-2-yl]-1-ethenyl-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

(A) [4-(tert-Butyl)-1,3-thiazol-2-yl]methanol

Ethyl thiooxamate (4.94 g, 37.1 mmol) was dissolved in anhydrous ethanol(250 ml), added with 1-bromopinacolone (4.99 ml, 37.1 mmol), andrefluxed by heating for 4 hours. The reaction mixture was concentrated,neutralized with saturated aqueous sodium hydrogencarbonate, andextracted with ethyl acetate. The organic layer was dried over sodiumsulfate, and then the solvent was evaporated under reduced pressure. Theresidue was dissolved in anhydrous ethanol (200 ml), added with sodiumhydroboride (2.10 g, 55.6 mmol), and stirred at room temperature for 16hours. The reaction solution was further added with sodium borohydride(500 mg, 13.2 mmol) and stirred at room temperature for 4 hours, andthen the solvent was evaporated under reduced pressure. The residue wascooled to −78° C., and the resulting white solid was taken by filtrationto obtain the title compound (2.77 g, 44%).

¹H-NMR (CDCl₃) δ: 1.33 (9H, s), 4.92 (2H, s), 6.86 (1H, s)

(B) 4-(tert-Butyl)-1,3-thiazol-2-carbaldehyde

[4-(tert-Butyl)-1,3-thiazol-2-yl]methanol (2.77 g, 16.2 mmol) obtainedin (A) was dissolved in methylene chloride (45 ml), added withpyridinium dichromate (12.2 g, 32.3 mmol), and stirred at roomtemperature for 13.5 hours. After insoluble solids were removed, thereaction solution was evaporated, and the residue was purified by silicagel column chromatography (dichloromethane) to obtain the title compound(2.71 g, 99%).

¹H-NMR (CDCl₃) δ: 1.40 (9H, s), 7.36 (1H, s), 9.98 (1H, s)

(C) tert-ButylN-(4-{2-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-hydroxyethyl}-2-pyridyl)-carbamate

tert-Butyl N-(4-methyl-2-pyridyl)carbamate (3.11 g, 14.9 mmol) wasdissolved in anhydrous tetrahydrofuran (50 ml), added with n-butyllithium (1.59 M in n-hexane, 19.7 ml, 31.3 mmol) at −78° C. under argonatmosphere, and stirred at room temperature for 1.5 hours. The reactionmixture was cooled to −78° C. again, then added with a solution of the4-(tert-butyl)-1,3-thiazol-2-carbaldehyde (2.30 g, 13.6 mmol) obtainedin (B) in anhydrous tetrahydrofuran (10 ml), and stirred for 30 minutes.The reaction mixture was added with saturated aqueous ammonium chloride,warmed to room temperature and extracted with chloroform. The organiclayer was dried over sodium sulfate, and the solvent was evaporatedunder reduced pressure. The residue was purified by silica gel columnchromatography (n-hexane:ethyl acetate=2:1→1:1, v/v) to obtain the titlecompound (2.29 g, 45%).

¹H-NMR (CDCl₃) δ: 1.32 (9H, s), 1.53 (9H, s), 3.08 (1H, dd, J=8.4,13.8Hz), 3.29 (1H, dd, J=4.2, 13.8), 5.23 (1H, dd, J=4.2, 8.4Hz), 6.83(1H, s), 6.85 (1H, dd, J=1.5, 5.1Hz), 7.67 (1H, s), 7.87 (1H, s), 8.13(1H, d, J=5.1Hz)

(D) tert-ButylN-(4-(E)-{2-[4-(tert-butyl)-1,3-thiazol-2-yl]-1-ethenyl}-2-pyridyl)-carbamate

The tert-butylN-(4-{2-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-hydroxyethyl}-2-pyridyl)carbamate(1.80 g, 4.77 mmol) obtained in (C) was dissolved in anhydroustetrahydrofuran (20 ml), added with triethylamine (2.33 ml, 16.7 mmol)and methanesulfonyl chloride (738 μl, 9.54 mmol), and stirred at 0° C.for 3.5 hours. After insoluble solids were removed, the reaction mixturewas washed with tetrahydrofuran, and the solvent was evaporated underreduced pressure. The residue was dissolved in toluene (30 ml), addedwith 1,8-diazabicyclo[5.4.0]undec-7-ene (1.07 ml, 7.15 mmol), andrefluxed by heating for 1 hour. The reaction mixture was concentrated,and the residue was purified by silica gel column chromatography(n-hexane:ethyl acetate=3:1→2:1, v/v) to obtain the title compound (1.23g, 72%).

¹H-NMR (CDCl₃) δ: 1.37 (9H, s), 1.55 (9H, s), 6.89 (1H, s), 7.08 (1H,dd, J=1.5, 5.4Hz), 7.29 (1H, d, J=16.4Hz), 7.51 (1H, d, J=16.4Hz), 8.12(1H, s), 8.09 (1H, s), 8.21 (1H, d, J=5.4Hz)

(E) 4-(E)-2-[4-(tert-Butyl)-1,3-thiazol-2-yl]-1-ethenyl-2-pyridinamine

The tert-butylN-(4-(E)-2-[4-(tert-butyl)-1,3-thiazol-2-yl]-1-ethenyl-2-pyridyl)-carbamate(585 mg, 1.63 mmol) obtained in (D) was dissolved in trifluoroaceticacid (15 ml) and stirred at room temperature for 1 hour. The reactionmixture was concentrated, neutralized with saturated aqueous sodiumhydrogencarbonate, and extracted with chloroform. The organic layer wasdried over sodium sulfate, and the solvent was evaporated under reducedpressure to obtain the title compound (422 mg, 100%).

¹H-NMR (CDCl₃) δ: 1.38 (9H, s), 6.62 (1H, s), 6.84 (1H, d, J=6.0Hz),6.93 (1H, s), 7.17 (1H, d, J=16.2Hz), 7.42 (1H, d, J=16.2Hz), 7.92 (1H,d, J=6.0Hz)

(F)8-(E)-2-[4-(tert-Butyl)-1,3-thiazol-2-yl]-1-ethenyl-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

Ethyl3-(dimethylamino)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-2-propenoate(150 mg, 0.453 mmol) was dissolved in propionic acid (2.0 ml), added tothe 4-(E)-2-[4-(tert-butyl)-1,3-thiazol-2-yl]-1-ethenyl-2-pyridinamine(98.0 mg, 0.378 mmol) obtained in (E), and refluxed by heating for 3hours. The reaction mixture was concentrated, and the residue waspurified by silica gel column chromatography (chloroform) to obtain thetitle compound (175 mg, 93%).

¹H-NMR (CDCl₃) δ: 1.40 (9H, s), 3.79 (3H, s), 5.81 (2H, s), 6.89 (2H, d,J=8.3Hz), 7.02 (1H, s), 7.41 (1H, d, J=16.2Hz), 7.42 (2H, d, J=8.3Hz),7.46 (1H, d, J=7.5Hz), 7.60 (1H, d, J=16.2Hz), 7.74 (1H, s), 9.21 (1H,d, J=7.5Hz), 9.21 (1H, s)

(G)8-(E)-2-[4-(tert-Butyl)-1,3-thiazol-2-yl]-1-ethenyl-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

The8-(E)-2-[4-(tert-butyl)-1,3-thiazol-2-yl]-1-ethenyl-3-(2H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(170 mg, 0.340 mmol) obtained in (F) was added with trifluoroacetic acid(4.0 ml) and stirred at 60° C. for 3 hours. After the reaction wascompleted, the trifluoroacetic acid was evaporated under reducedpressure, and the residue was neutralized with 0.1 N sodium hydroxideand phosphate buffer (pH 7-8) and extracted with chloroform. The organiclayer was dried over sodium sulfate, and the solvent was evaporatedunder reduced pressure. The yellow solid deposited from the residue wascollected by filtration to obtain the title compound (20.0 mg, 15%).

¹H-NMR (DMSO-d₆) δ: 1.35 (9H, s), 7.47 (1H, s), 7.67 (1H, d, J=16.1Hz),8.03 (1H, d, J=16.1Hz), 8.04 (1H, d, J=1.9Hz), 8.13 (1H, d, J=1.9Hz),9.11 (1H, d, J=7.3Hz), 9.17 (1H, s)

EI-MS; m/z: 380 (M⁺+1)

Example 112(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl(E)-3-(2-hydroxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate(17 mg) was added with dimethylformamide (3 ml), diisopropylethylamine(500 ml) and 3-(iodomethyl)tetrahydrofuran (50 ml), and then the mixturewas stirred at 80° C. for 16 hours. The reaction mixture was added withethyl acetate and saturated brine, and the organic layer was furtherwashed twice with saturated brine and dried over magnesium sulfate.After the solvent was evaporated under reduced pressure, the residue waspurified by silica gel column chromatography to obtain the titlecompound (14 mg).

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=7.1Hz), 1.52 (9H, s), 1.75 (1H, m),2.16 (1H, m), 2.82 (1H, m), 3.07 (1H, m), 3.26 (2H, m), 3.39 (2H, m),3.71 (1H, m), 3.80 (1H, m), 3.92 (2H, m), 4.42 (2H, m), 6.74 (1H, s),7.02 (1H, m), 7.13 (1H, d, J=15.8Hz), 7.34 (1H, s), 7.91 (1H, d,J=15.8Hz), 8.99 (1H, d, J=7.3Hz)

(B)(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(10 mg) obtained in (A) was dissolved in methylene chloride (1 ml) andformic acid (1 ml), and then the mixture was stirred for 5 hours. Afterthe solvent was evaporated under reduced pressure, the residue waspurified by silica gel column chromatography to obtain the titlecompound (14 mg).

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.6Hz), 1.79 (1H, m), 2.16 (1H, m),2.83 (1H, m), 3.07 (1H, m), 3.26 (2H, m), 3.39 (2H, m), 3.73 (2H, m),3.81 (1H, m), 3.93 (2H, m), 4.42 (2H, m), 6.73 (1H, s), 7.02 (1H, m),7.15 (1H, d, J=15.8Hz), 7.33 (1H, s), 8.05 (1H, d, J=15.8Hz), 8.99 (1H,d, J=6.5Hz)

Example 1138-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-4-one

(A)(E)-2-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-1-ethenylcyanide

(E)-2-{2-Hydroxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-1-ethenylcyanide(100 mg) was added with dimethylformamide (5 ml), diisopropylethylamine(500 ml) and 3-(iodomethyl)tetrahydrofuran (200 ml), and then themixture was stirred at 80° C. for 2 hours and further stirred overnightat room temperature. After the reaction mixture was distributed betweenchloroform and water, the organic layer was dried over magnesiumsulfate. After the solvent was evaporated under reduced pressure, theresidue was purified by silica gel column chromatography to obtain thetitle compound (51 mg).

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.8Hz), 1.78 (1H, m), 2.16 (1H, m),2.79 (1H, m), 3.05 (1H, m), 3.29 (2H, m), 3.38 (2H, m), 3.71 (1H, m),3.82 (1H, m), 3.93 (2H, m), 4.42 (2H, m), 6.74 (1H, s), 6.68 (1H, d,J=16.3Hz), 7.07 (1H, d, J=7.3Hz), 7.37 (1H, s), 7.64 (1H, d, J=16.3Hz),8.97 (1H, d; J=7.3Hz)

(B)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-4-one

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.8Hz), 1.80 (1H, m), 2.18 (1H, m),2.86 (1H, m), 3.08 (1H, m), 3.27 (2H, m), 3.42 (2H, m), 3.80 (2H, m),3.96 (2H, m), 4.47 (2H, m), 6.76 (1H, s), 7.04 (1H, m), 7.34 (1H, s),7.90 (1H, d, J=16.3Hz), 8.04 (1H, d, J=16.3Hz), 8.99 (1H, d, J=7.0Hz)

Example 114(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(2-pyridylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

¹H-NMR (CD₃OD-CDCl₃) δ: 1.42 (6H, d, J=6.8Hz), 3.29 (1H, m), 3.40 (2H,m), 3.77 (2H, m), 5.96 (2H, s), 7.18 (1H, d, J=15.9Hz), 7.37 (1H, m),7.44 (1H, s), 7.80 (1H, s), 8.02 (1H, d, J=15.9Hz), 8.07 (1H, m), 8.31(1H, m), 8.68 (1H, m), 8.92 (1H, m), 9.04 (1H, d, J=7.3Hz)

Example 115(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-pyridylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD-CDCl₃) δ: 1.42 (6H, d, J=6.8Hz), 3.29 (1H, m), 3.45 (2H,m), 3.83 (2H, m), 5.85 (2H, s), 7.07 (1H, d, J=15.9Hz), 7.39 (1H, d,J=7.3Hz), 7.46 (1H, s), 7.78 (1H, s), 7.93 (1H, d, J=15.9Hz), 8.18 (1H,m), 8.82 (2H, m), 9.01 (1H, d, J=7.1Hz), 9.24 (1H, s)

Example 116(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-pyridylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.35 (6H, d, J=6.8Hz), 3.29 (1H, m), 3.37 (2H, m),3.70 (2H, m), 5.97 (2H, s), 7.16 (1H, d, J=15.9Hz), 7.38 (1H, d,J=7.0Hz), 7.52 (2H, s), 8.08 (1H, d, J=15.9Hz), 8.20 (2H, m), 8.88 (2H,m), 9.06 (1H, d, J=7.0Hz)

Example 117(E)-3-{8-[(E)-2-(4-Isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A)8-[(E)-2-(4-Isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-.a]pyrimidin-4-one

2-Hydroxy-8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-4-one(210 mg, 0.670 mmol) was suspended in dimethylformamide (6.0 ml), addedwith diisopropylethylamine (600 μl) and 3-(iodomethyl)tetrahydrofuran(334 μl), and stirred overnight at 80° C. The reaction solution wasfurther added with diisopropylethylamine (600 al) and3-(iodomethyl)tetrahydrofuran (334 μl) and stirred overnight at 80° C.The reaction mixture was concentrated, and the residue was purified bysilica gel column chromatography(chloroform:methanol=100:0→100:1→100:2→100:5, v/v) to obtain the titlecompound (277 mg, quantitative).

(B) tert-Butyl(E)-3-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

Dimethylformamide (3.0 ml) was added with phosphorus oxychloride (187μl, 2.02 mmol) under ice cooling and stirred at room temperature for 30minutes. The mixture was added to8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-4-one(277 mg) obtained in (A) and dissolved in dimethylformamide (3.0 ml)under ice cooling, and then the mixture was stirred at room temperaturefor 4 hours. The reaction mixture was added with saturated aqueoussodium hydrogencarbonate and extracted with chloroform. The organiclayer was dried over magnesium sulfate, and the solvent was evaporatedunder reduced pressure. The residue was suspended in anhydroustetrahydrofuran (6.0 ml) and anhydrous N,N-dimethylformamide (3.0 ml),added with (tert-butoxycarbonylmethylene)triphenylphosphorane (505 mg,1.34 mmol) and stirred at 50° C. for 16 hours. The reaction mixture wasconcentrated, and then the residue was purified by silica gel columnchromatography (n-hexane:ethyl acetate, 5:1→3:1→2:1→1:1, v/v) to obtainthe title compound (215 mg, 61%).

¹H-NMR (CDCl₃) δ: 1.36 (6H, d, J=6.8Hz), 1.53 (9H, s), 1.80 (1H, dt,J=7.3, 12.8Hz), 2.12-2.21 (1H, m), 2.80-2.87 (1H, m), 3.12-3.19 (1H, m),3.73 (1H, dd, J=5.4, 8.8Hz), 3.81 (1H, dd, J=7.7, 15.2Hz), 3.91-3.97(2H, m), 4.44-4.47 (2H, m), 6.99 (1H, s), 7.16 (1H, d, J=16.3Hz), 7.31(1H, d, J=1.8, 7.5Hz), 7.39 (1H, d, J=16.2Hz), 7.48 (1H, d, J=1.5Hz),7.54 (1H, d, J=16.2Hz), 7.92 (1H, d, J=16.3Hz), 9.04 (1H, d, J=7.5Hz)

ESI-MS; m/z: 524 (M⁺+1)

(C)(E)-3-{8-[(E)-2-(4-Isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-butyl(E)-3-{8-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-2-(tetrahydro-3-furanylmethoxy)-4H-pyrido[1,2-a(pyrimidin-3-yl}-2-propenoate(101 mg, 0.193 mmol) obtained in (B) was dissolved in 1,4-dioxane (1.6ml), added with 4 N hydrochloric acid in 1,4-dioxane (3.2 ml), and thenthe mixture was stirred at room temperature for 16.25 hours. Thereaction solution was concentrated, added with saturated aqueous sodiumhydrogencarbonate, neutralized with phosphate buffer (pH 7-8), and thenextracted with a mixture of chloroform/methanol (10:1, v/v). The organiclayer was dried over magnesium sulfate, and then the solvent wasevaporated under reduced pressure. The residue was purified by thinlayer chromatography (chloroform:methanol=20:1, v/v) to obtain the titlecompound (77.1 mg, 86%).

¹H-NMR (CDCl₃+CD₃OD) δ: 1.36 (6H, d, J=6.8Hz), 1.17-1.85 (1H, m),2.13-2.22 (1H, m), 2.82-2.89 (1H, m), 3.13-3.19 (1H, m), 3.74 (1H, dd,J=5.4, 8.8Hz), 3.82 (1H, dd, J=7.7, 15.5Hz), 3.93-3.98 (2H, m),4.44-4.52 (2H, m), 7.01 (1H, s), 7.16 (1H, d, J=15.9Hz), 7.36-7.41 (2H,m), 7.50 (1H, s), 7.57 (1H, d, J=16.1Hz), 8.03 (1H, d, J=15.9Hz), 9.03(1H, d, J=7.3Hz)

ESI-MS; m/z: 468 (M⁺+1)

Example 1188-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-3-(1H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

(A)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-3-[(4-methoxybenzyl)tetrazol-5-yl]-4H-pyrido[1,2-a]pyrimidin-4-one

4-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-aminopyridine (50 mg) anddiethyl 2-[1-(4-methoxybenzyl)tetrazol-5-yl]malonate (141 mg) wereheated to 150° C. in bromobenzene with stirring for 4 hours. After thesolvent was evaporated, the residue was purified by silica gel columnchromatography to obtain the title compound (73 mg) as cream solid.

¹H-NMR (CDCl₃) δ: 1.22 (d, J=6.6Hz, 6H), 3.02 (m, 1H), 3.20-3.40 (m,4H), 3.70 (s, 3H), 5.80 (s, 2H), 6.70 (s, 1H), 6.80 (bs, 2H), 7.00 (s,1H), 7.30-7.50 (m, 3H), 9.00 (bs, 1H)

MS(−), m/z, 502 (M−H⁺)

(B)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-tosyloxy-3-[(4-methoxybenzyl)tetrazol-5-yl]-4H-pyrido[1,2-a]pyrimidin-4-one

The8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-3-[(4-methoxybenzyl)-tetrazol-5-yl]-4H-pyrido[1,2-a]pyrimidin-4-one(73mg) obtained in (A) was dissolved in methylene chloride (2 ml), addedwith dimethylaminopyridine (5 mg), triethylamine(40 ml) andp-toluenesulfonyl chloride (83 mg) at 0° C., and then the mixture wasstirred at room temperature for 6 hours. The reaction solution wasdirectly purified by silica gel column chromatography to obtain thetitle compound (61 mg, 64%) as pale yellow powder.

¹H-NMR (CDCl₃) δ: 1.22 (d, J=6.6Hz, 6H), 2.42 (8, 3H), 3.05 (m, 1H),3.25-3.32 (m, 4H), 3.80 (s, 3H), 5.75 (s, 2H), 6.75 (s, 1H), 6.85 (d,J=7.2Hz, 2H), 7.15 (d, J=7.5Hz, 1H), 7.24 (d, J=7.2Hz, 2H), 7.35 (d,J=7.2Hz, 2H), 7.42 (8, 1H), 7.90 (d, J=7.2Hz, 2H), 9.01 (d, J=7.5Hz, 1H)

(C)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl)-2-morpholino-3-(1-p-methoxybenzyl-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

The8-1-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-tosyloxy-3-[(4-methoxybenzyl)-tetrazol-5-yl]-4H-pyrido[1,2-a]pyrimidin-4-one(61 mg) obtained in (B) was dissolved in tetrahydrofuran (1 ml), addedwith morpholine (40 ml) at room temperature, and stirred overnight atthe same temperature. After the solvent was evaporated under reducedpressure, the residue was purified by silica gel column chromatographyto obtain the title compound (31 mg, 59%).

¹H-NMR (CDCl₃) δ: 1.28 (d, J=6.6Hz, 6H), 3.06 (m, 1H), 3.30-3.70 (m,12H), 3.78 (s, 3H), 5.76 (s, 2H), 6.72 (s, 1H), 6.77 (d, J=7.5Hz, 1H),6.86 (d, J=7.2Hz, 2H), 7.14 (s, 1H), 7.36 (d, J=7.2Hz, 2H), 8.88 (m,J=7.5Hz, 1H)

(D)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-3-(1H-1,2,3,4-tetrazol-5-yl)4H-pyrido[1,2-a]pyrimidin-4-one

The 8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl-3-2-morpholino-3-(1-p-methoxybenzyl-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one(10 mg) obtained in (C) was dissolved in trifluoroacetic acid (1 ml) andadded with anisole (50 ml), and then stirred at room temperature for 4hours. After the solvent was evaporated, the residue was purified bysilica gel column chromatography to obtain the title compound (7 mg) asyellow powder.

¹H-NMR (CDCl₃) δ: 1.25 (d, 6H), 3.04 (m, 1H), 3.22 (t, 2H), 3.38 (t,2H), 3.66 (m, 4H), 3.82 (m, 4H), 6.73 (s, 1H), 6.89 (m, 1H), 7.22 (m,1H), 8.83 (d, 1)H

MS (ES−) m/z 451 (M⁺−1)

In the following Examples 119 to 121, synthesis was performed in thesame manner as in Example 118.

Example 1198-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-pyridylmethoxy)-3-(1H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

¹H-NMR (CDCl₃) δ: 1.21 (d, 6H), 2.98 (m, 1H), 3.25 (t, 2H), 3.36 (t,2H), 5.73 (s, 2H), 6.71 (s, 1H), 7.16 (d, 1H), 7.31 (m, 1H), 7.42 (s,1H), 8.10 (d, 1H),8.41 (d, 1H), 8.69 (s, 1H), 8.99 (d, 1H)

MS (ES+) m/z 475 (M⁺+1); MS (ES−) m/z 473 (M⁺−1)

Example 1208-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-dimethylamino-3-(1H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

¹H-NMR (CD₃OD) δ: 1.24 (d, 6H), 2.95 (s, 6H), 3.01 (m, 1H), 3.21 (t,2H), 3.41 (t, 2H), 6.95 (s, 1H), 7.00 (m, 1H), 7.23 (s, 1H), 8.80 (d,1H)

MS (ES+) m/z 512 (M⁺+1); MS (ES−) m/z 510 (M⁺−1)

Example 1218-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(4-cyanopiperidino)-3-(1H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

¹H-NMR (CDCl₃) δ: 1.33 (d, 6H), 2.02 (m, 2H), 2.10 (m, 2H), 2.95 (m,1H), 3.20 (m, 4H), 3.45 (m, 2H), 3.62 (m, 1H), 3.79 (m, 2H), 6.82 (s,1H), 6.90 (d, 1H), 7.18 (s, 1H), 8.85 (d, 1H)

MS (ES−) m/z 476 (M⁺+1); 474 (M⁺−1)

Example 1224-Isopropyl-2-(2-(3-[([(4-methylphenyl)sulfonyl]aminocarbonyl)amino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-8-yl)ethyl)-1,3-thiazole

(A) tert-ButylN-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-ylcarbamate

4-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-aminopyridine (300 mg) andmethyl 2-(t-butoxycarbonyl)amino-3-dimethylaminopropenoate (445 mg) wereadded with xylene (2 ml) and heated at 140° C. for 6.5 hours withstirring. After the solvent was evaporated, the residue was purified bysilica gel column chromatography to obtain the title compound (200 mg)as yellow powder.

¹H-NMR (CDCl₃) δ: 1.21 (d, J=6.9Hz, 6H), 3.01 (m, 1H), 3.22 (t, J=7.8Hz,2H), 3.37 (t, J=7.8Hz, 2H), 6.72 (s, 1H), 6.96 (d, J=7.2Hz, 1H), 7.25(s, 1H), 7.47 (s, 1H), 8.83 (d, J=7.5Hz, 1H), 9.14 (s, 1H)

MS(+), m/z, 415 (M+H⁺), 829 (2M+H⁺)

(B)3-Amino-8-[2-(4-isopropyl-1,3thiazol-2yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4one

The tert-butylN-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2a]pyrimidin-3-ylcarbamate(200 mg) obtained in (A) was treated with 2 ml of trifluoroacetic acidat room temperature for 1 hour, and the solvent was evaporated underreduced pressure. The residue was dissolved in ethyl acetate, washedwith saturated sodium hydrogencarbonate and dried over sodium sulfate.The solvent was evaporated under reduced pressure, and the residue waspurified by silica gel column chromatography to obtain the titlecompound (quantitative) as yellow powder.

¹H-NMR (CDCl₃) δ: 1.28 (d, J=6.9Hz, 6H), 3.06 (m, 1H), 3.23 (t, J=7.8Hz,2H), 3.39 (t, J=7.8Hz, 2H), 6.75 (s, 1H), 6.95 (d, J=7.5Hz, 1H), 7.50(s, 1H), 7.47 (s, 1H), 7.94 (s, 1H), 8.80 (d., J=7.5Hz, 1H)

MS(+), m/z, 315 (M+H⁺)

(C)4-Isopropyl-2-(2-3-[([(4-methylphenyl)sulfonyl]aminocarbonyl)amino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-8-ylethyl)-1,3-thiazole

The3-amino-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]-pyrimidin-4-one(44 mg) obtained in (B) and p-toluenesulfonyl isocyanate (42 mg) weredissolved in toluene (0.5 ml) and refluxed by heating for 2.5 hours.After the solvent was evaporated, the residue was purified by silica gelcolumn chromatography to obtain the title compound (33 mg) as whitepowder.

¹H-NMR (CDCl₃) δ: 1.26 (d, 6H), 2.41 (s, 3H), 3.05 (m, 1H), 3.25 (t,2H), 3.39 (t, 2H), 6.73 (s, 1H), 7.19 (dd, 1H), 7.31 (d, 1H), 7.56 (s,1H), 7.96 (d, 1H), 8.71 (s, 1H), 9.04 (d, 1H), 9.39 (s, 1 H)

MS (ES+) m/z 411 (M⁺+1)

Example 123(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) Ethyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

4-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-aminopyridine (200 mg),diethyl 4-dimethylaminomethyleneglutaconate (265 mg) was added withbromobenzene (2 ml) and heated at 120° C. for 1 hour and at 160° C. for4 hours with stirring. The residue was purified by silica gel columnchromatography to obtain the title compound (20 mg) as white powder.

¹H-NMR (CDCl₃) δ: 1.20-1.40 (m, 9H), 3.05 (m, 1H), 3.32 (t, J=6.6Hz,2H), 3.40 (t, J=6.6Hz, 2H), 4.27 (q, J=7.2Hz, 2H), 6.73 (s, 1H), 7.11(d, J=7.2Hz, 1H), 7.18 (d, J=15.9Hz, 1H), 7.51 (s, 1H), 7.66 (d,J=15.9Hz, 1H), 8.45 (s, 1H), 9.08 (s, 1H)

MS(+), m/z, 398 (M+H⁺), 420 (M+Na⁺), MS(−), m/z, 396 (M−H⁺)

(B)(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-44H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The ethyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(15 mg) obtained in (A) was dissolved in tetrahydrofuran (1 ml) andmethanol (0.5 ml), added with a solution of 8 mg of lithium hydroxidedissolved in 0.5 ml of water, and stirred at room temperature for 3.5hours, and the solvent was concentrated under reduced pressure. Theresidue was distributed between ether and water, and the aqueous layerwas separated, made pH 3 with hydrochloric acid, extracted with ethylacetate, and dried over sodium sulfate. After the solvent wasevaporated, the residue was purified by silica gel column chromatographyto obtain the title compound.

¹H-NMR (CDCl₃) δ: 1.21 (d, 6H), 2.41 (s, 3H), 3.00 (m, 1H), 3.25 (t,2H), 3.34 (t, 2H), 6.67 (s, 1H), 7.06-7.19 (m, 2H), 7.48 (s, 1H), 7.68(d, J=16Hz, 1H), 8.40 (s, 1H), 9.03 (d, J=7.5Hz, 1H)

MS (ES+) m/z 370 (M⁺+1); MS (ES−) m/z 368 (M⁺−1)

Example 1242-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yloxy}aceticacid

(A) Ethyl2-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yloxy}acetate

4-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-aminopyridine (100 mg) andethyl 2-ethoxycarbonylmethoxy-3-methoxy-3-dimethylaminopropenoate (150mg) were heated at 140° C. in xylene (1 ml) for 7 hours with stirring.After the solvent was evaporated under reduced pressure, the residue waspurified by silica gel column chromatography to obtain the titlecompound (41 mg).

¹H-NMR(CDCl_(3,) 300MHz) δ: 1.20-1.30 (m, 9H), 3.04 (m, 1H), 3.23 (t,J=7.8Hz, 2H), 3.40 (t, J=7.8Hz, 2H), 4.25 (q, J=7.2Hz, 2H), 4.86 (s,2H), 6.72 (s, 1H), 6.98 (d, J=7.5Hz, 1H), 7.45 (s, 1H), 8.29 (s, 1H),8.90 (d, J=7.5Hz, 1H)

MS(+), m/z, 401 (M+H⁺)

(B)2-(8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yloxy)aceticacid

The ethyl2-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yloxy}acetate(41 mg) obtained in (A) was dissolved in tetrahydrofuran (1 ml) andmethanol (300 ml), added with a solution of lithium hydroxide (5 mg)dissolved in water (300 ml), and then the mixture was stirred at roomtemperature for 1 hour, and the solvent was concentrated under reducedpressure. The residue was distributed between ether and water, and theaqueous layer was separated and made pH 3 with hydrochloric acid, andthen extracted with ethyl acetate. The organic layer was dried oversodium sulfate. After the solvent was evaporated, the residue waspurified by silica gel column chromatography to obtain the titlecompound (quantitative).

¹H-NMR (DMSO-d₆) δ: 1.23 (d, 6H), 3.00 (m, 1H), 3.21 (t, 2H), 3.40 (t,2H), 4.54 (s, 2H), 6.96 (s, 1H), 7.18 (d, 1H), 7.38 (s, 1H), 8.12 (s,1H), 8.85 (d, 1H)

MS (ES−) m/z 372 (M⁺−1)

Example 1255-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2,3-dihydro-1,3,4-oxadiazol-2-one

Methyl8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carboxylate(22 mg) and hydrazine (4 ml) were refluxed by heating in methanol (3 ml)for 2.5 hours under nitrogen atmosphere. The reaction mixture wasfurther added with hydrazine (10 ml) and refluxed by heating for 2 days.After the reaction mixture was cooled, insoluble solids were collectedby filtration and suspended in methylene chloride (5 ml). The suspensionwas added with diphosgene (7 ml) and stirred for 30 minutes. Insolublesolids were removed by filtration, and the reaction mixture wasconcentrated to obtain the title compound (11 mg).

¹H-NMR (CD₃OD) δ: 1.36 (s, 3H), 1.39 (s, 3H), 3.22 (m, 1H), 3.53 (m,2H), 3.77 (m, 2H); 7.58 (s, 1H); 7.75 (d, 1H), 7.91 (s, 1H), 8, 84 (s,1H), 9.31 (d, 1H)

MS (ES+) m/z 384 (M⁺+1); MS (ES−) m/z 382 (M⁺−1)

Example 126(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoicacid

(A) tert-Butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoate

tert-Butyl 2-(diethylphosphono)propionate (70 mg) was dissolved intetrahydrofuran(5 ml), added with sodium hydride (60% in oil, 40 mg),and stirred for 10 minutes. The reaction mixture was added with8-(2-(4-isopropyl-1,3-thiazol-2-yl)ethyl)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde(56 mg), stirred for 10 minutes and added with acetic acid (0.2 ml), andthen distributed between ethyl acetate and saturated aqueous sodiumhydrogencarbonate. The organic layer was concentrated, and the residuewas purified by silica gel column chromatography to obtain the titlecompound (70 mg) as yellow oil.

¹H-NMR (CDCl₃) δ: 1.27 (d, 6H), 1.51 (s, 9H), 2.07 (s, 3H), 3.05 (m,1H), 3.3-3.5 (m, 4H), 6.72 (s, 1H), 7.07 (d, 1H), 7.52 (s, 1H), 7.68 (s,1H), 8.41 (s, 1H), 9.00 (s, 1H)

(B)(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoicacid

The tert-butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-44H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoate(70 mg) obtained in (A) was dissolved in trifluoroacetic acid (1 ml) andstirred at room temperature for 1 hour, and then the solvent wasevaporated under reduced pressure. The residue was purified by silicagel column chromatography to obtain the title compound (21 mg).

¹H-NMR (CDCl₃+CD₃OD) δ: 1.25 (d, 6H), 2.10 (s, 3H), 3.04 (m, 1H), 3.28(t, 2H), 3.35-3.45 (m, with CD₃OD), 6.74 (s, 1H), 7.15 (d, 1H), 7.59 (s,1H), 7.79 (s, 1H), 8.45 (s, 1H), 9.01 (d, 1H)

Example 127(E)-2-(3-Chloropropyl)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.28 (d, 6H), 2.10 (m, 2H), 2.70 (m, 2H), 3.05 (m,1H), 3.30 (t, 2H), 3.40 (t, 2H), 3.60 (m, 2H), 6.75 (s, 1H), 7.10 (d,1H), 7.54 (s, 1H), 7.94 (s, 1H), 8.51 (s, 1H), 9.03 (d, 1H)

MS (ES+) m/z 446 (M⁺+1); MS (ES−) m/z 444 (M⁺−1)

Example 128(E)-3-{8-[2-(4-Isopropyl)-1,3-thiazol-2-yl]ethyl}-4-oxo-2-phenyl-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A)2-Trifluoromethanesulfonlyloxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one

2-Hydroxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one(400 mg) and DMAP (310 mg) were dissolved in methylene chloride (8 ml),added with trifluoromethanesulfonic anhydride (427 ml) at −78° C., andthen the mixture was stirred overnight while the reaction temperaturewas gradually returned to room temperature. The reaction mixture wasadded with 0.2 M hydrochloric acid (50 ml) and extracted with methylenechloride, and the organic layer was dried over sodium sulfate. After thesolvent was evaporated, the residue was purified by silica gel columnchromatography to obtain the title compound (499 mg, 88%).

¹H-NMR (CDCl₃) δ: 1.22 (d, J=7.5Hz, 6H), 3.10 (m, 1H), 3.25-3.40 (m,4H), 6.10 (s, 1H), 6.75 (s, 1H), 7.18 (d, J=7.5Hz, 1H), 7.55 (s, 1H),8.95 (d, J=7.5Hz, 1H)

(B)2-Phenyl-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one

The2-trifluoromethanesulfonyloxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one(82 mg) obtained in (A), phenylboronic acid (45 mg), palladiumtetrakistriphenylphosphine (11 mg), potassium bromide (24 mg) andpotassium carbonate (38 mg) were stirred at 85° C. overnight in dioxane(4 ml) under a nitrogen flow. The reaction mixture was cooled and addedwith water (20 ml) and extracted with ethyl acetate, and then theorganic layer was dried over sodium sulfate. The solvent was evaporatedunder reduced pressure, and the residue was purified by silica gelcolumn chromatography to obtain the title compound (65 mg, 94%) as whitepowder.

¹H-NMR (CDCl₃) δ: 1.22 (d, J=7.5Hz, 6H), 3.00-3.10 (m, 1H), 3.30-3.50(m, 4H), 6.72 (s, 1H), 6.85 (s, 1H), 6.99 (d, J=7.5Hz, 1H), 7.46-7.56(m, 5H), 8.20-8.40 (m, 1H), 8.96 (d, J=7.5Hz, 1H)

(C) tert-Butyl(E)-3-{2-phenyl-4-oxo-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

Dimethylformamide (1 ml) and phosphorus oxychloride (25 ml) were mixedunder ice cooling and stirred at room temperature for 30 minutes. Themixture was added with a solution of the2-phenyl-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one(65 mg) obtained in (B) and dissolved in dimethylformamide (1 ml) andstirred at room temperature for 1 hour and at 95° C. for 1.5 hours on anouter bath. The reaction mixture was cooled and then added with ethylacetate and hexane. Then, the organic layer was washed with saturatedaqueous sodium hydrogencarbonate and water and dried over magnesiumsulfate, and the solvent was evaporated under reduced pressure. Theresidue was added with tetrahydrofuran (2.5 ml) and dimethylformamide(0.5 ml), further added with(tert-butoxycarbonylmethylidene)triphenylphosphorane (240 mg), andstirred at 80° C. for 10 hours. The solvent was evaporated under reducedpressure, and the residue was purified by silica gel columnchromatography to obtain the title compound (18 mg).

¹H-NMR (CDCl₃) δ: 1.22 (d, J=7.5Hz, 6H), 3.00-310 (m, 1H), 3.30-3.50 (m,4H), 6.82 (s, 1H), 6.99 (d, J=7.5Hz, 1H), 7.40-7.66 (m, 7H), 8.05-8.10(m, 1H), 8.95 (d, J=7.5Hz, 1H)

(D)(E)-3-{8-[2-(4-Isopropyl)-1,3-thiazol-2-yl]ethyl}-4-oxo-2-phenyl-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-butyl(E)-3-{2-phenyl-4-oxo-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(18 mg) obtained in (C) was added with trifluoroacetic acid (1 ml) andstirred at room temperature for 2 hours. The solvent was evaporatedunder reduced pressure, and the residue was purified by silica gelcolumn chromatography to obtain the title compound (15 mg) as yellowpowder.

¹H-NMR (CDCl₃) δ: 1.25 (d, 6H), 3.05 (m, 1H), 3.28 (t, 2H), 3.39 (t,2H), 6.75 (s, 1H), 7.12 (d, 1H), 7.39 (d, J=16Hz, 1H), 7.50 (m, 3H),7.58 (m, 3H), 7.72 (d, J=16Hz, 1H), 9.05 (d, 1H)

Example 129(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-pyridyl)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-pyridyl)-4H-pyrido[1,2-a]pyrimidin-4-one

2-Trifluoromethanesulfonyloxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one(499mg), pyridine-3-boronic acid 1,3-propanediol cyclic ester (364 mg),palladium tetrakistriphenylphosphine (65 mg), potassium bromide (146 mg)and potassium carbonate (231 mg) were added with dioxane(8 ml) andstirred overnight at 85° C. under nitrogen atmosphere. The reactionmixture was cooled and then added with ethyl acetate. Insoluble solidswere removed by filtration, and the solvent of the filtrate wasevaporated. The residue was purified by silica gel column chromatographyto obtain the title compound (400 mg).

¹H-NMR (CDCl₃) δ: 1.18 (d, J=7.2Hz, 6H), 2.98 (m, 1H), 3.05-3.30 (m,4H), 6.66 (s, 1H), 6.75 (s, 1H), 7.02 (dd, J=7.5, 1.8Hz, 1H), 7.38 (dd,J=7.5, 7.8Hz, 1H), 7.49 (d, J=1.2Hz, 1H), 8.30 (dt, J=7.5, 1.8Hz, 1H),8.55 (dd, J=7.5, 1.2Hz, 1H), 8.86 (d, J=7.8Hz, 1H), 9.11 (d, J=1.2Hz,1H).

(B) Methyl(E)-3-(8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-pyridyl)-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

Phosphorus oxychloride (367 ml) was added to dimethylformamide (5 ml)under ice cooling and stirred at the same temperature for 10 minutes.The mixture was added with the8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-pyridyl)-4H-pyrido[1,2-a]pyrimidin-4-one(336 mg) obtained in (A) and stirred at 95° C. for 1.5 hours. Themixture was cooled, then slowly added with sodium carbonate andextracted with ethyl acetate. The organic layer was dried over sodiumsulfate, and the solvent was evaporated under reduced pressure. Theresidue was purified by silica gel column chromatography to obtainyellow powder (58 mg). The resulting powder was dissolved intetrahydrofuran (2.5 ml) and dimethylformamide (0.5 ml), added withmethyl (triphenylphosphoranylidene)acetate (240 mg) and stirred at 90°C. for 10 hours. After the solvent was evaporated under reducedpressure, the residue was purified by silica gel column chromatographyto obtain the title compound (19 mg) as yellow powder.

¹H-NMR (CDCl₃) δ: 1.22 (d, J=7.5Hz, 6H), 3.05 (m, 1H), 3.30-3.50 (m,4H), 3.75 (s, 3H), 6.88 (s, 1H), 7.15 (dd, J=7.2, 2;1Hz, 1H), 7.40-7.60(m, 4H), 7.93 (dt, J=7.8, 1.8Hz, 1H), 8.75 (dd, J=5.1, 1.5Hz, 1H), 8.87(d, J=1.8Hz, 1H), 9.08 (d, J=7.5Hz, 1H)

(C)(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-pyridyl)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The methyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(3-pyridyl)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoateobtained in (B) was dissolved in tetrahydrofuran (2 ml) and methanol(0.5 ml), added with a solution (0.5 ml) containing lithium hydroxide(5.5 mg) and stirred at room temperature for 1 hour. The solvent wasevaporated under reduced pressure, and the residue was distributedbetween water and diethyl ether. The aqueous layer was separated, madepH 4-5 with hydrochloric acid, and then extracted with ethyl acetate.The organic layer was dried over sodium sulfate, and the solvent wasevaporated. The residue was purified by silica gel column chromatographyto obtain the title compound (6.9 mg) as yellow powder.

¹H-NMR (CDCl₃) δ: 1.18 (d, 6H), 2.95 (m, 1H), 3.21 (t, 2H), 3.32 (t,2H), 6.67 (s, 1H), 7.15 (d, 1H), 7.23 (d, J=13Hz, 1H), 7.41 (d, J=13Hz,1H), 7.48 (s, 1H), 7.55 (m, 1H), 8.01 (m, 1H), 8.65 (m, 1H), 8.76 (s,1H), 8.98 (d, 1H)

MS (ES+) m/z 447 (M⁺+1); MS (ES−) m/z 445 (M⁺−1)

Example 130(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-2-(4-pyridyl)-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.25 (d, 6H), 3.02 (m, 1H), 3.2-3.5 (m, with CHD₂OD),6.87 (s, 1H), 7.33 (d, J=16Hz, 1H), 7.38 (m, 1H), 7.45 (d, J=16Hz, 1H),7.59 (m, 2H), 7.72 (s, 1H), 8.72 (m, 2H), 9.13 (d, 1H)

MS (ES+) m/z 447 (M⁺+1); MS (ES−) m/z 445 (M⁺−1)

Example 131(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoic acid

(A) Ethyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoate

Under nitrogen atmosphere, triethyl 2-phosphonopropionate (155 mg, 0.65mmol) was dissolved in tetrahydrofuran (2 ml), added with n-butyllithium (1 M, 0.65 ml) at −78° C., and stirred at the same temperaturefor 20 minutes. The reaction mixture was added with a solution of8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (50 mg) dissolved in tetrahydrofuran (1 ml). The coolingbath was removed, and the reaction mixture was warmed to roomtemperature and stirred overnight at room temperature. The reactionmixture was added with saturated aqueous sodium hydrogencarbonate andextracted with ethyl acetate. The organic layer was dried over magnesiumsulfate, and the solvent was evaporated under reduced pressure. Theresidue was purified by silica gel column chromatography to obtain thetitle compound (14 mg).

¹H-NMR (CDCl₃) δ: 1.20-1.30 (m, 9H), 1.79 (s, 3H), 2.95 (m, 1H), 3.11(t, J=7.5Hz, 2H), 3.29 (t, J=7.5Hz, 2H), 3.48 (t, J=4.8Hz, 4H), 3.66 (t,J=4.8Hz, 4H), 4.05-4.15 (m, 2H), 6.66 (s, 1H), 6.75 (d, J=7.5Hz, 1H),7.12 (s, 1H), 7.45 (s, 1H), 8.75 (d, J=7.5Hz, 1H)

(B)(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoic acid

The ethyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoate(14 mg) obtained in (A) was dissolved in tetrahydrofuran (4 ml) andmethanol (1 ml), added with a solution of lithium hydroxide (1.4 mg) inwater (1 ml) and stirred at room temperature for 1.5 hours. The reactionmixture was added with water and washed with ether, and then the aqueouslayer was made acidic with diluted hydrochloric acid and extracted withethyl acetate. The organic layer was dried over magnesium sulfate, andthe solvent was evaporated under reduced pressure. The residue waspurified by silica gel column chromatography to obtain the titlecompound (4.4 mg) as yellow powder.

¹H-NMR (CDCl₃) δ: 1.24 (d, 6H), 1.86 (s, 3H), 3.05 (m, 1H), 3.19 (t,2H), 3.38 (t, 2H), 3.58 (m, 4H), 3.74 (m, 4H), 6.74 (s, 1H), 6.82 (d,1H), 7.20 (s, 1H), 7.63 (s, 1H), 8.83 (d, 1H)

MS (ES+) m/z 469 (M⁺+1); MS (ES−) m/z 467 (M⁺−1)

Example 132(E)-2-Methyl-3-(8-{2-[4-(1-methylcyclopropyl)-1,3-thiazol-2-yl]ethyl}-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

¹H-NMR (CDCl₃) δ: 0.75 (m, 2H), 1.14 (m, 2H), 1.42 (s, 3H), 1.88 (s,3H), 3.18 (t, 2H), 3.34 (t, 2H), 3.59 (m, 4H), 3.76 (m, 4H), 6.73 (s,1H), 6.81 (d, 1H), 7.22 (s, 1H), 7.63 (s, 1H), 8.82 (d, 1H)

MS (ES+) m/z 481

Example 133(E)-3-{8-[2-(4-tert-Butyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methyl-2-propenoicacid

¹H-NMR (CDCl₃) δ: 1.35 (s, 9H), 1.85 (s, 3H), 3.20 (t, 2H), 3.36 (t,2H), 3.58 (m, 4H), 3.75 (m, 4H), 6.73 (s, 1H), 6.81 (d, 1H), 7.20 (s,1H), 7.61 (s, 1H), 8.81 (d, 1H)

MS (ES+) m/z 483 (M⁺+1); MS (ES−) m/z 481 (M⁺−1)

Example 134(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-aminopyrrolidino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-methyl-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.20 (d, 6H), 1.59 (s, 3H), 1.95 (m, 1H), 2.23 (m,1H), 2.98 (m, 1H), 3.10 (m, 2H), 3.35 (m, 2H), 3.43 (m, 1H), 3.62 (m,2H), 3.75 (m, 2H), 6.88 (m, 2H), 7.08 (s, 1H), 7.36 (s, 1H), 8.62 (d,1H)

MS (ES+) m/z 468 (M⁺+1)

Example 135(Z)-2-Fluoro-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) Ethyl(Z)-2-fluoro-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

2-Fluoro-2-phosphonoacetic acid triethyl ester (384 mg) was dissolved intetrahydrofuran (3 ml) and dimethylformamide (1.5 ml), added withn-butyl lithium (1.6 M, 1 ml) at −78° C. under nitrogen atmosphere, andthen the mixture was stirred at the same temperature for 20 minutes. Thereaction mixture was added with a solution of8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4-oxo-4H-pyrido[1,2-a]-pyrimidine-3-carbaldehyde(108 mg) dissolved in tetrahydrofuran (2 ml). The reaction temperaturewas gradually raised to room temperature, and the reaction mixture wasstirred overnight at the same temperature. The reaction mixture wasadded with water and extracted with methylene chloride, and the organiclayer was dried over magnesium sulfate. Then, the solvent was evaporatedunder reduced pressure. The residue was purified by silica gel columnchromatography to obtain the title compound (35 mg) as a mixture of (E)-and (Z)-isomers.

¹H-NMR (CDCl₃) δ: 1.20-1.30 (m, 9H), 3.05 (m, 1H), 3.20-3.30 (m, 4H),4.05-4.15 (m, 2H), 6.70-6.80 (m, 2H), 7.14 (d, J=7.5Hz, 1H), 7.41 (s,1H), 9.00-9.05 (m, 1H)

MS(+), m/z, 382 (M+H⁺); MS(−), m/z, 380 (M−H⁺)

(B) Ethyl(Z)-2-fluoro-3-(8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

The ethyl2-fluoro-3-(8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate(35 mg) obtained in (A) was dissolved in methylene chloride (2 ml),added with triethylamine (35 ml) and tosyl chloride (46 mg) under icecooling, and then the mixture was stirred overnight at room temperature.The reaction mixture was added with morpholine (49 ml) and furtherstirred for 4 hours. After the solvent was evaporated under reducedpressure, the residue was purified by silica gel column chromatographyto obtain yellow powder (28 mg). This was dissolved in methylenechloride (1 ml), added with a trace amount of iodine, and then themixture was stirred at room temperature for 30 minutes, and the solventwas evaporated to obtain the title compound.

¹H-NMR (CDCl₃) δ: 1.20-1.30 (m, 9H), 3.05 (m, 1H), 3.20 (t, J=7.8Hz,2H), 3.36 (t, J=7.8Hz, 2H), 3.55-3.80 (m, 8H), 4.20-4.35 (m, 2H),6.70-6.82 (m, 2H), 7.19 (d, J=7.5Hz, 1H), 7.35 (d, J=36.9Hz, 1H),8.75-8.85 (m, 1H)

(C)(Z)-2-Fluoro-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The ethyl(Z)-2-fluoro-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(28 mg) obtained in (B) was dissolved in tetrahydrofuran (2 ml) andmethanol (0.5 ml), added with a solution of lithium hydroxide(5 mg) inwater (0.5 ml), and then the mixture was stirred at room temperature for1 hour. The reaction mixture was added with water and washed with ether.Then, the aqueous layer was made acidic with diluted hydrochloric acidand extracted with ethyl acetate. The organic layer was dried overmagnesium sulfate, and the solvent was evaporated under reducedpressure. The residue was purified by silica gel column chromatographyto obtain the title compound (26 mg) as yellow powder.

¹H-NMR (CD₃OD) δ: 1.24 (d, 6H), 3.03 (m, 1H), 3.18 (t, 2H), 3.37 (t,2H), 3.62 (m, 4H), 3.72 (m, 4H), 6.88-7.0 (m, 3H), 7.18 (s, 1H), 8.72(d, 1H)

MS (ES+) m/z 473 (M⁺+1); MS (ES−) m/z 471 (M⁺−1)

Example 136(Z)-2-Fluoro-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.2-1.4 (m, 7H), 1.52 (m, 2H), 1.80 (m, 1H), 2.0 (m,1H), 3.0 (m, 2H), 3.2 (t, 2H), 3.38 (t, 2H), 3.65 (m, 1H), 3.9 (m, 1H),4.15 (m, 1H), 6.97 (m, 2H), 7.15 (d, J=36Hz, 1H), 7.15 (s, 1H), 8.66 (d,1H)

Example 137(Z)-2-Fluoro-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-aminocarbonylmorpholino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.24 (d, 6H), 3.0 (m, 2H), 3.2 (m, 3H), 3.38 (t, 2H),3.68 (m, 1H), 4.0 (m, 3H), 4.46 (d, 1H), 6.97 (s, 1H), 7.03 (d, 1H),7.13 (s, 1H), 7.19 (d, J=36Hz, 1H), 7.25 (s, 1H), 8.75 (d, 1H)

MS (ES−) m/z 514 (M⁺−1)

Example 138(Z)-2-Fluoro-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-(3-cyanomorpholino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

¹H-NMR (CDCl₃) δ: 1.22 (d, 6H), 3.0 (m, 1H), 3.15 (m, 2H), 3.25 (m, 4H),3.70-4.0 (m, 4H), 4.26 (dd, 1H), 6.72 (s, 1H), 6.81 (d, 1H), 6.85 (d,J=36Hz, 1H), 7.16 (s, 1H), 8.72 (d, 1H)

MS (ES−) m/z 496 (M⁺−1)

Example 139(Z)-2-Fluoro-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-(4-aminomethylcarbonylpiperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.22 (d, 6H), 3.02 (m, 1H), 3.21 (t, 2H), 3.39 (t,2H), 3.53 (m, 2H), 3.65 (m, 6H), 3.98 (s, 2H), 6.98 (s, 1H), 7.05 (d,1H), 7.22 (s, 1H), 7.26 (d, J=37Hz, 1H), 8.77 (d, 1H)

MS (ES+) m/z 529 (M⁺+1); MS (ES−) m/z 527 (M⁺−1)

Example 140(Z)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methoxy-2-propenoicacid

(A) Ethyl(Z)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methoxy-2-propenoate

2-Methoxy-2-phosphonoacetic acid triethyl ester (92 mg) was dissolved intetrahydrofuran (1 ml), added with n-butyl lithium (1.6 M, 0.23 ml) at−78° C. under nitrogen atmosphere, and then the mixture was stirred atthe same temperature for 30 minutes. The reaction mixture was added witha solution of8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde(50 mg) dissolved in tetrahydrofuran (1 ml). The reaction temperaturewas gradually raised to room temperature, and the reaction mixture wasstirred overnight at the same temperature. The reaction mixture wasadded with water and extracted with methylene chloride. The organiclayer was dried over magnesium sulfate, and the solvent was evaporatedunder reduced pressure. The residue was purified by silica gel columnchromatography to obtain the title compound.

¹H-NMR (CDCl₃) δ: 1.20-1.30 (m, 9H), 3.05 (m, 1H), 3.15-3.35 (m, 4H),3.50 (s, 3H), 3.55-3.70 (m, 8H), 4.05-4.15 (m, 2H), 6.70-6.80 (m, 2H),7.14 (s, 1H), 7.21 (s, 1H), 8.78 (d, J=7.5Hz, 1H)

(B)(Z)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-44H-pyrido[1,2-a]pyrimidin-3-yl}-2-methoxy-2-propenoicacid

The ethyl(Z)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-methoxy-2-propenoateobtained in (A) was dissolved in tetrahydrofuran (2 ml) and methanol(0.5 ml), added with a solution of lithium hydroxide (5 mg) in water(0.5 ml), and then the mixture was stirred at room temperature for 2.5hours. The reaction mixture was added with water and washed with ether,and then the aqueous layer was made acidic with diluted withhydrochloric acid and extracted with ethyl acetate. The organic layerwas dried over magnesium sulfate, and the solvent was evaporated underreduced pressure to obtain the title compound (6 mg, 10% for the twosteps) as yellow powder.

¹H-NMR (CD₃OD) δ: 1.23 (d, 6H), 3.02 (m, 1H), 3.19 (t, 2H), 3.38 (t,2H), 3.55 (s, 3H), 3.62 (m, 4H), 3.70 (m, 4H), 6.95 (s, 1H), 6.99 (m,2H), 7.18 (s, 1H), 8.74 (d, 1H)

MS (ES+) m/z 485 (M⁺+1); MS (ES−) m/z 483 (M⁺−1)

Example 1415-(1-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}methylidene)-1,3-thiazolidine-2,4-dione

8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde(18 mg) and 2,4-thiazolidinedione (53 mg) were added with benzene (10ml), piperidine (one drop) and acetic acid (two drops) and refluxed byheating for 3 hours in a vessel provided with a Dean-Stark trap. Afterthe solvent was evaporated under reduced pressure, the residue waspurified by silica gel column chromatography to obtain the titlecompound (17 mg) as yellow powder.

¹H-NMR (CDCl₃) δ: 1.26 (d, 6H), 3.01 (m, 1H), 3.22 (t, 2H), 3.38 (t,2H), 3.63 (m, 4H), 3.80 (m, 4H), 6.73 (s, 1H), 6.87 (d, 1H), 7.23 (s,1H), 7.78 (s, 1H), 8.80 (d, 1H)

MS (ES−) m/z 510 (M⁺−1)

Example 142(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-methoxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-methoxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(20 mg) and triethylamine (63 ml) were dissolved in dimethylformamide(1.5 ml) and stirred at room temperature for 20 minutes under nitrogenatmosphere. The reaction mixture was added with dimethyl sulfate (4.3ml) and stirred in the dark for 2 days. The reaction mixture was furtheradded with dimethyl sulfate (4.3 ml) and stirred for 2 days in the samemanner. The reaction mixture was added with water and extracted withethyl acetate. The organic layer was washed with 1% aqueous solution oflithium chloride and dried over magnesium sulfate, and the solvent wasevaporated under reduced pressure. The residue was purified by silicagel column chromatography to obtain the title compound (6.7 mg) asyellow powder. The starting material (7.6 mg) was also recovered.

¹H-NMR (CDCl₃): 1.28 (s, 3H), 1.29 (s, 3H), 1.51 (s, 9H), 3.07 (m, 1H),3.21 (m, 2H), 3.42 (m, 2H), 4.06 (s, 3H), 6.74 (s, 1H), 7.01 (d, 1H),7.11 (d, 1H), 7.37 (s, 1H), 7.94 (d, 1H), 9.00 (d, 1H)

(B) (E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-methoxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

The tert-butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-methoxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(6.7 mg) obtained in (A) was treated with trifluoroacetic acid (0.5 ml)in the dark for 30 minutes, and then the solvent was evaporated. Theresidue was added with methanol and methylene chloride, and insolublematters were collected by filtration to obtain the title compound (7.2mg).

¹H-NMR (CDCl₃): 1.30 (s, 3H), 1.32 (s, 3H), 3.11. (m, 1H), 3.31 (m, 2H),3.45 (m, 2H), 4.07 (s, 3H), 6.77 (s, 1H), 7.05 (d, 1H), 7.18 (d, 1H),7.39 (s, 1H), 8.10 (d, 1H), 9.02 (d, 1H)

MS (ES−): 398

Example 143(Z)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-methoxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-methoxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid (1 mg) was dissolved in CDCl₃ (0.5 ml) and irradiated with lightfrom a fluorescent lamp for 19 hours. The solvent was evaporated toobtain the title compound (1 mg).

¹H-NMR (CDCl₃) δ: 1.28 (s, 3H), 1.29 (s, 3H), 3.12 (m, 1H), 3.38 (m,2H), 3.61 (m, 2H), 4.06 (s, 3H), 6.19 (d, 1H), 6.80 (d, 1H), 6.90 (s,1H), 7.05 (d, 1H), 7.40 (d, 1H), 7.50 (s, 1H), 9.04 (d, 1H)

MS (ES−): 398

Example 144(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

tert-Butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(15 mg) was treated with formic acid (1 ml) in the dark for 1 hour, andadded with toluene. The solvent was evaporated under reduced pressure toobtain the title compound (22 mg).

¹H-NMR (DMSO-d₆) δ: 1.18 (s, 3H), 1.21 (s, 3H), 2.99 (m, 1H), 3.23-3.40(m, 4H), 6.82 (d, 1H), 7.10 (s, 1H), 7.20 (s, 1H), 7.42 (d, 1H), 7.85(d, 1H), 8.91 (d, 1H)

MS (ES−): 384

Example 145(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-9-methoxy-2-morpholino-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) 3-Methoxy-4-methyl-2-nitropyridine

3-Hydroxy-4-methyl-2-nitropyridine (5 g) was dissolved indimethylformamide (50 ml), added with cesium carbonate (11.6 g) andmethyl iodide (13.7 g), and then the mixture was stirred overnight atroom temperature. The reaction mixture was added with ethyl acetate andhexane, washed with water and dried over magnesium sulfate, and then thesolvent was evaporated under reduced pressure to obtain the titlecompound (quantitative).

¹H-NMR (CDCl₃) δ: 2.30 (s, 3H), 3.78 (s, 3H), 7.27 (d, J=4.8Hz, 1H),7.98 (d, J=4.8Hz, 1H)

(B) 2-Amino-3- methoxy-4-methylpyridine

The 3-methoxy-4-methyl-2-nitropyridine (1 g) obtained in (A) wasdissolved in methanol (50 ml), added with 5% Pd/C (200 mg), and stirredat a pressure of 40 psi for 2 hours under hydrogen atmosphere. After thecatalyst was removed by filtration, the solvent was evaporated underreduced pressure to obtain the title compound (850 mg) as yellow oil.

¹H-NMR (CDCl₃) δ: 2.20 (s, 3H), 3.70 (s, 3H), 4.6 (s, 2H), 6.45 (d,J=4.8Hz, 1H), 7.85 (d, J=4.8Hz, 1H)

(C) 2-(tert-Butoxycarbonylamino)-3-methoxy-4-methylpyridine

The 2-amino-3-methoxy-4-methylpyridine (850 mg) obtained in (B) wasdissolved in tert-butanol (10 ml), added with di-tert-butyl dicarbonate(2 g), and stirred at room temperature for 72 hours. After the solventwas evaporated under reduced pressure, the residue was purified bysilica gel column chromatography to obtain the title compound (1.1 g).

¹H-NMR (CDCl₃) δ: 1.52 (s, 9H), 2.27 (s, 3H), 3.75 (s, 3H), 4.6 (s, 1H),6.78 (d, J=4.8Hz, 1H), 8.05 (d, J=4.8Hz, 1H)

(D) tert-ButylN-4-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-3-methoxy-2-pyridylcarbamate

The 2-(tert-butoxycarbonylamino)-3-methoxy-4-methylpyridine (2.84 g)obtained in (C) was dissolved in anhydrous tetrahydrofuran (50 ml) andadded dropwise with n-butyl lithium (1.6 M, 19 ml) at −78° C. undernitrogen atmosphere. Then, the reaction temperature was raised to roomtemperature. The reaction mixture was cooled to −78° C. again, addeddropwise with a solution of 2-bromomethyl-4-isopropylthiazole (3.94 g)dissolved in tetrahydrofuran (10 ml) and stirred at the same temperaturefor 1 hour. Then, the reaction mixture was added with water andextracted with ethyl acetate. The organic layer was dried over sodiumsulfate, and the solvent was evaporated under reduced pressure. Theresidue was purified by silica gel column chromatography to obtain thetitle compound (4.1 g).

¹H-NMR (CDCl₃) δ: 1.20 (d, J=6.9Hz, 6H), 1.45 (s, 9H), 2.95-3.20 (m,5H), 3.67 (s, 3H), 6.60 (s, 1H), 6.70 (d, J=4.8Hz, 1H), 8.05 (d,J=4.8Hz, 1H)

(E) 2-Amino-4-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-3-methoxypyridine

The tert-butylN-4-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-3-methoxy-2-pyridylcarbamate(4.1 g) obtained in (D) was added with trifluoroacetic acid (20 ml) atroom temperature and stirred overnight. Then, the reaction mixture wasadded with 50 ml of water and 5 ml of 6 M hydrochloric acid and washedwith ether. The aqueous layer was carefully added with sodiumhydrogencarbonate to make pH of the aqueous layer weakly alkaline, andthen extracted with ethyl acetate. The organic layer was dried oversodium sulfate, and the solvent was evaporated under reduced pressure toobtain the title compound (2.7 g).

¹H-NMR (CDCl₃) δ: 1.20 (d, J=6.9Hz, 6H), 3.00-3.30 (m, 5H), 3.70 (s,3H), 4.6 (s, 2H), 6.50 (s, 1H), 6.70 (d, J=4.8Hz, 1H), 7.75 (d, J=4.8Hz,1H)

(F) 2-Hydroxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one

The 2-amino-4-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-3-methoxypyridine(2.35 g) obtained in (E) and bis-2,4,6-trichlorophenyl malonate (4.3 g)were refluxed by heating for 1 hour in toluene (25 ml), and then thesolvent was evaporated under reduced pressure. The residue was purifiedby silica gel column chromatography to obtain the title compound (2.8g).

¹H-NMR (CDCl₃) δ: 1.28 (d, J=6.9Hz, 6H), 2.95-3.05 (m, 1H), 3.30-3.40(m, 4H), 3.95 (s, 3H), 5.35 (s, 1H), 6.70 (s, 1H), 7.00 (d, J=7.2Hz,1H), 8.82 (d, J=7.2Hz, 1H)

(G)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-9-methoxy-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one

The2-hydroxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-9-methoxy-4H-pyrido[1,2-a]pyrimidin-4-one(1.77 g) obtained in (F) was dissolved in methylene chloride (40 ml),added with triethylamine (1.5 ml) and tosyl chloride (1.96 g) under icecooling, and then the mixture was stirred at room temperature. Afterdisappearance of the starting material was observed, the reactionmixture was added with morpholine (2.2 ml) and stirred overnight. Afterthe solvent was evaporated under reduced pressure, the residue waspurified by silica gel column chromatography to obtain the titlecompound (0.99 g) as yellow powder.

¹H-NMR (CDCl₃) δ: 1.28 (d, J=6.9Hz, 6H), 3.00-3.10 (m, 1H), 3.20 (t,J=7.2Hz, 2H), 3.30 (t, J=7.2Hz, 2H), 3.65-3.80 (m, 8H), 4.00 (s, 3H),5.60 (s, 1H), 6.65-6.70 (m, 2H), 8.80 (d, J=7.2Hz, 1H)

(H)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-9-methoxy-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde

Dimethylformamide (2 ml) was added with phosphorus oxychloride (0.6 ml)under ice cooling and stirred at room temperature for 20 minutes. Thismixture was cooled again with ice, and added with a solution of the8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-9-methoxy-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one(0.99 g) obtained in (G) in methylene chloride (10 ml). The reactionmixture was stirred at room temperature for 3 hours, added with water,then added with saturated aqueous sodium hydrogencarbonate and extractedwith methylene chloride. After the organic layer was dried over sodiumsulfate, the solvent was evaporated and the residue was purified bysilica gel column chromatography to obtain the title compound (1.54 g).

¹H-NMR (CDCl₃) δ: 1.28 (d, J=6.9Hz, 6H), 3.00-3.10 (m, 1H), 3.20 (t,J=7.2Hz, 2H), 3.30 (t, J=7.2Hz, 2H), 3.70-3.85 (m, 8H), 3.95 (s, 3H),6.75 (d, J=7.5Hz, 1H), 8.00 (s, 1H), 8.55 ((d, J=7.5Hz), 10.05 (s, 1H)

(I) tert-Butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-9-methoxy-2-morpholino-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

The8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-9-methoxy-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde(300 mg) obtained in (H) was dissolved in tetrahydrofuran (4 ml) anddimethylformamide (1 ml), added with(tert-butoxycarbonylmethylene)triphenylphosphorane (767 mg), and stirredat 80° C. for 15 hours. After the solvent was evaporated, the residuewas purified by silica gel column chromatography to obtain the titlecompound (209 mg) as yellow powder.

¹H-NMR (CDCl₃) δ: 1.25 (d, J=6.9Hz, 6H), 1.5 (s, 9H), 3.00-3.10 (m, 1H),3.20-3.35 (m, 4H), 3.60-3.85 (m, 8H), 4.00 (s, 3H), 6.70 (s, 1H), 6.80(d, J=7.5Hz, 1H), 7.05 (d, J=15.3Hz, 1H), 7.50 (d, J=15.3Hz, 1H), 8.65(d, J=7.5Hz, 1H)

(J)(E)-3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-9-methoxy-2-morpholino-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-9-methoxy-2-morpholino-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(209 mg) obtained in (1) was dissolved in trifluoroacetic acid (2 ml)and stirred at room temperature for 30 minutes. The solvent wasevaporated under reduced pressure to obtain the title compound (180 mg)as yellow powder.

¹H-NMR (CDCl₃) δ: 1.22 (d, 6H), 3.00 (m, 1H), 3.16 (m, 2H), 3.25 (t,2H), 3.55 (m, 4H), 3.78 (m, 4H), 3.98 (s, 3H), 6.68 (s, 1H), 6.94 (d,J=16Hz, 1H), 7.58 (d, J=16Hz, 1H), 8.58 (d, 1H)

MS (ES+) m/z 485 (M⁺+1), MS (ES−) m/z 483 (M⁺−1)

Example 1465-((Z)-1-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-ylmethylidene)-1,3-thiazolidine-2,4-dione

(A) Methyl8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate

4-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-aminopyridine (497 mg) anddimethyl methoxymethylenemalonate (425 mg) were dissolved in methylenechloride, and stirred at 90° C. for 2 hours while the solvent wasevaporated. The reaction mixture was added with propionic acid (0.5 ml)and heated at 160° C. for 10 hours with stirring. The reaction mixturewas cooled, then added with ethyl acetate, washed with saturated sodiumhydrogencarbonate and dried over sodium over sodium sulfate. The solventwas evaporated under reduced pressure, and the residue was purified bysilica gel column chromatography to obtain the title compound (416 mg).

¹H-NMR (CDCl₃) δ: 1.28 (8, 3H), 1.31 (s, 3H), 3.06 (m, 1H), 3.35 (m,2H), 3.40 (m, 2H), 3.99 (s, 3H), 6.75 (8, 1H), 7.20 (d, 1H), 7.60 (s,1H), 9.07 (s, 1H), 9.19 (d, 1H)

(B)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehydeThe methyl8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-carboxylate(660 mg) obtained in (A) was dissolved in tetrahydrofuran (30 ml), addeddropwise with diisopropylaluminum hydride (1 M solution intetrahydrofuran, 9.2 ml) at −78° C., and then the mixture was stirredfor 2 hours at the same temperature. The reaction mixture was added withsaturated aqueous ammonium chloride (1 ml), then added with 12% aqueoushydrochloric acid, and stirred at room temperature for 1 hour. Afterinsoluble solids were removed by filtration through a Celite layer, thesolvent of the filtrate was evaporated, and the residue was dissolved inmethylene chloride. This solution was added with active manganesedioxide (1.2 g) and stirred at room temperature for 16 hours. Then,insoluble matters were removed by filtration to obtain the titlecompound (390 mg) as yellow powder.

¹H-NMR (CDCl₃) δ: 1.27 (d, 6H), 3.05 (m, 1H), 3.3-3.5 (m, 4H), 6.74 (s,1H), 7.25 (d, 1H), 7.63 (s, 1H), 8.87 (s, 1H), 9.16 (d, 1H), 10.36 (s,1H)

(C)5-((Z)-1-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-ylmethylidene)-1,3-thiazolidine-2,4-dione

The8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde(70 mg) obtained in (B) and 2,4-thiazolidinedione (360 mg) were addedwith benzene (10 ml), piperidine (one drop) and acetic acid (two drops)and refluxed by heating for 1 hour in a vessel provided with aDean-Stark trap. After the solvent was evaporated under reducedpressure, the residue was purified by silica gel column chromatographyto obtain the title compound (62 mg) as yellow powder.

¹H-NMR (CDCl₃+CD₃OD) δ: 1.22 (d, 6H), 3.00 (m, 1H), 3.2-3.4 (m, withCD₃OD), 6.72 (s, 1H), 7.18 (d, 1H), 7.51 (s, 1H), 7.86 (s, 1H), 8.42 (s,1H), 8.98 (d, 1H)

MS (ES+) m/z 427 (M⁺+1), MS (ES−) m/z 425 (M⁺−1)

Example 1473-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-4,5-dihydro-1,2,4-oxadiazol-5-one

(A)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide

A mixture of methyl8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate(400 mg), concentrated aqueous ammonia (6 ml) and methanol (9 ml) wasstirred overnight. Insoluble solids were collected by filtration toobtain the title compound (117 mg). The reaction mixture wasconcentrated and the residue was purified by silica gel columnchromatography to further obtain the title compound (69 mg).

¹H-NMR (CDCl₃) δ: 1.29 (s, 3H), 1.31 (s, 3H), 3.09 (m, 1H), 3.39 (m,2H), 3.48 (m, 2H), 5.73 (brs, 1H), 6.79 (s, 1H), 7.29 (d, 1H), 7.66 (s,1H), 8.71 ( brs, 1H), 9.14 (d, 1H), 9.30 (s, 1H)

MS (ES+) m/z 343 (M⁺+1), MS (ES−) m/z 341 (M⁺−1)

(B)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbonitrile

A mixture of the8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxamide(186 mg) obtained in (A), p-toluenesulfonyl chloride (207 mg) andpyridine (0.18 ml) was stirred overnight in methylene chloride. Themixture was added with triethylamine (0.2 ml), further stirred for 2days, and diluted with methylene chloride. This mixture was washed withsaturated aqueous sodium hydrogencarbonate and saturated brine, anddried over sodium sulfate, and the solvent was evaporated under reducedpressure. The residue was purified by silica gel column chromatographyto obtain the title compound (98 mg). The starting material was alsorecovered (73 mg).

¹H-NMR (CDCl₃) δ: 1.29 (s, 3H), 1.31 (s, 3H), 3.09 (m, 1H), 3.37 (m,2H), 3.49 (m, 2H), 6.79 (s, 1H), 7.31 (d, 1H), 7.65 (s, 1H), 8.58 (s,1H), 9.09 (d, 1H)

MS (ES+) m/z 325 (M⁺+1), MS (ES−) m/z 323 (M⁺−1)

(C) 3-{8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-4,5-dihydro-1,2,4-oxadiazol-5-one

The8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbonitrile(35 mg) obtained in (B), hydroxylamine (9 mg) and triethylamine (28 ml)were refluxed overnight by heating in ethanol. After the reactionmixture was cooled, the solvent was evaporated under reduced pressure,and the residue was purified by silica gel column chromatography toobtain an oxim compound (22 mg). This compound (11 mg),1,1′-carbodiimidazole (5 mg) and pyridine (2 ml) were dissolved intetrahydrofuran (0.5 ml), refluxed by heating for 45 minutes, andstirred overnight at room temperature. The reaction mixture wasconcentrated, and the residue was purified by silica gel columnchromatography to obtain the title compound (1.6 mg).

¹H-NMR (CDCl₃) δ: 1.26 (s, 3H), 1.30 (s, 3H), 3.08 (m, 1H), 3.40 (m,4H), 6.75 (s, 1H), 7.18 (brm, 1H), 7.70 (brs, 1H), 9.08 (brm, 1H)

MS (ES+) m/z 384 (M⁺+1), MS (ES−) m/z 382 (M⁺−1)

Example 1482-Hydroxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-3-(1H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

2-Hydroxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-3-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4H-pyrido[1,2-a]pyrimidin-4-one(30 mg) was added with anisole (0.15 ml) and trifluoroacetic acid (1 ml)and stirred at room temperature for 20 hours. After the solvent wasevaporated under reduced pressure, the residue was added with methanoland toluene and the solvents were evaporated again under reducedpressure. The residue was added with methanol and methylene chloride,and the deposited crystals were collected by filtration to obtain thetitle compound (15 mg).

¹H-NMR (CD₃OD) δ: 1.25 (s, 3H), 1.28 (s, 3H), 3.04 (m, 1H), 3.35 (m,2H), 3.46 (m, 2H), 7.04 (s, 1H), 7.31 (s, 1H), 7.45 (d, 1H), 9.11 (d,1H)

MS (ES+) m/z 384 (M⁺+1)

Example 1498-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-methoxy-3-(1H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

(A)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-methoxy-3-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4H-pyrido[1,2-a]pyrimidin-4-one

2-Hydroxy-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-3-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4H-pyrido[1,2-a]pyrimidin-4-one(24 mg) was dissolved in dimethylformamide (1.5 ml), added withtriethylamine (1.6 ml) and dimethyl sulfate (22 ml) and stirredovernight at room temperature. The reaction mixture was further addedwith dimethyl sulfate (5 ml), stirred for 4 hours, and then added withwater and extracted with ethyl acetate. After the organic layer wasdried over sodium sulfate, the solvent was evaporated under reducedpressure, and the residue was purified by silica gel columnchromatography to obtain the title compound (5 mg).

¹H-NMR (CDCl₃) δ: 1.28 (s, 3H), 1.33 (s, 3H), 3.09 (m, 1H), 3.29 (m,2H), 3.44 (m, 2H), 3.79 (s, 3H), 4.03 (s, 3H), 5.79 (s, 2H), 6.79 (s,1H), 6.88 (d, 2H), 7.02 (d, 1H), 7.41 (d, 2H), 9.02 (d, 1H)

MS (ES+) m/z 518 (M⁺+1), MS (ES−) m/z 516 (M⁺−1)

(B)8-[2-(4-Isopropyl-1,3-thiazol-2-yl)ethyl]-2-methoxy-3-(1H-1,2,3,4-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one

The8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-methoxy-3-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-4H-pyrido[1,2-a]pyrimidin-4-one(5 mg) obtained in (A) was added with trifluoroacetic acid (0.3 ml) andanisole (0.1 ml) and stirred for 2 days. The solvent was evaporatedunder reduced pressure, and the residue was added with hexane and ethylacetate. Insoluble solids were collected by filtration and dried toobtain the title compound (1.2 mg).

¹H-NMR (CDCl₃) δ: 1.30 (s, 3H), 1.32 (s, 3H), 3.12 (m, 1H), 3.37 (m,2H), 3.49 (m, 2H), 4.29 (s, 3H), 6.79,(s, 1H), 7.53 (s, 1H), 9.10 (d,1H)

MS (ES+) m/z 398 (M⁺+1), MS (ES−) m/z 396 (M⁺−1)

Example 150(E)-3-(2-Carboxymethylthio-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-(2-(diphenoxyphosphoryl)oxy]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)-ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl(E)-3-{8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(80 mg) was suspended in methylene chloride (40 ml), added withtriethylamine (0.13 ml) and diphenylphosphoryl chloride (0.15 ml), andthen the mixture was stirred at room temperature for 16 hours. Thereaction mixture was washed with saturated aqueous sodiumhydrogencarbonate, 5% aqueous hydrochloric acid and saturated brine, andthen dried over sodium sulfate and the solvent was evaporated underreduced pressure. The residue was purified by silica gel columnchromatography to obtain the title compound (104 mg).

¹H-NMR (CDCl₃) δ: 1.26 (d, 6H), 1.51 (s, 9H), 3.05 (m, 1H), 3.2-3.4 (m,4H), 6.73 (s, 1H), 7.1-7.5 (m, 13H), 7.75 (d, J=15.8Hz, 1H), 8.96 (d,1H)

(B) tert-Butyl(E)-3-{2-(tert-butoxycarbonylmethylthio)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

The tert-butyl(E)-3-(2-[(diphenoxyphosphoryl)oxy]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(62 mg) obtained in (A) was dissolved in dimethylformamide (1 ml) andadded dropwise with a solution of lithium sulfide in ethanol (0.1 g/ml)until the starting material disappeared. Separately, tert-butylbromoacetate (0.04 ml) was dissolved in dimethylformamide (1 ml), addedwith sodium iodide (69 mg), and then stirred at room temperature for 40minutes. These two of solutions were mixed and stirred at roomtemperature for 2 hours. The reaction mixture was distributed betweenethyl acetate and water, and the organic layer was dried. The solventwas evaporated, and the residue was purified by silica gel columnchromatography to obtain the title compound (19 mg).

¹H-NMR (CDCl₃) δ: 1.29 (d, 6H), 1.45 (s, 9H), 1.53 (s, 9H), 3.06 (m,1H), 3.2-3.4 (m, 4H), 3.93 (s, 2H), 6.73 (s, 1H), 6.99 (d, 1H), 7.24 (d,J=15.6Hz, overlapped with CHCl₃, 1H), 7.32 (s, 1H), 7.78 (d, J=15.6Hz,1H), 8.95 (d, 1H)

(C)(E)-3-{2-Carboxymethylthio-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-butyl(E)-3-{2-(tert-butoxycarbonylmethylthio)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(19 mg) obtained in (B) was dissolved in methylene chloride (2 ml),added with triethylsilane (0.5 ml) and trifluoroacetic acid (0.5 ml),and then the mixture was stirred at room temperature for 5 hours. Afterthe solvent was evaporated under reduced pressure, the residue waspurified by silica gel column chromatography to obtain the titlecompound (4 mg) as yellow powder.

¹H-NMR (CDCl₃) δ: 1.18 (d, 6H), 2.96 (m, 1H), 3.15 (m, 2H), 3.25 (m,1H), 3.91 (d, 2H), 6.69 (s, 1H), 6.99 (d, 1H), 7.15 (d, J=15Hz, 1H),7.31 (s, 1H), 7.77 (d, J=15Hz, 1H), 8.83 (d, J=7Hz, 1H)

MS (ES+) m/z 460 (M⁺+1), MS (ES−) m/z 458 (M⁺−1)

Example 151(E)-3-{2-Methylthio-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

¹H-NMR (CDCl₃) δ: 1.21 (d, 6H), 2.56 (s, 3H), 3.00 (m, 1H), 3.19 (m,2H), 3.31 (m, 2H), 6.69 (s, 1H), 6.99 (d, 1H), 7.19 (d, J=15Hz, 1H),7.36 (s, 1H), 7.83 (d, J=15Hz, 1H), 8.87 (d, J=7Hz, 1H)

Example 152(E)-3-{2-Aminocarbonylmethylthio-8-[2-(4-isopropyl-i,3-thiazol-2-yl)-ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

¹H-NMR (CDCl₃+CD₃OD) δ: 1.23 (d, 6H), 3.02 (m, 1H), 3.1-3.5 (m, withCD₃OD), 3.88 (d, 2H), 6.74 (s, 1H), 7.06 (d, 1H), 7.15 (d, J=16Hz, 1H),7.41 (s, 1H), 7.74 (d, J=16Hz, 1H), 8.89 (d, J=8Hz, 1H)

MS (ES+) m/z 458 (M⁺+1)

Example 153(E)-3-{2-[2-(Aminoethylthiomethyl)-3-pyridylthio]-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (CD₃OD) δ: 1.22 (d, 6H), 3.02 (m, 1H), 3.15-3.4 (m, with CHD₂OD),6.95 (s, 1H), 7.02 (s, 1H), 7.28 (d, 1H), 7.32 (d, J=16Hz, 1H), 7.52(dd, 1H), 8.02 (d, J=16Hz, 1H), 8.10 (d, 1H), 8.72 (m, 1H), 8.98 (d, 1H)

Example 1541-Ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro[1,8]naphthylidine-3-carboxylicacid

(A) Ethyl1-ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro[1,8]naphthylidine-3-carboxylate

[(4-Isopropyl-1,3-thiazol-2-yl)methyl](triphenyl)phosphonium bromide(723 mg, Chem. Pharm. Bull., 1977, 25, 349-352) was suspended intetrahydrofuran (20 ml), added dropwise with n-butyl lithium (1.6 M, 1.2mmol) at −20° C. under nitrogen atmosphere, and stirred at the sametemperature for 20 minutes. The reaction mixture was added with asolution of ethyl1-ethyl-7-formyl-4-oxo-1,4-dihydro-[1,8]naphthylidine-3-carboxylate (316mg) dissolved in tetrahydrofuran, and stirred for 2 hours. The reactionmixture was added with saturated aqueous ammonium chloride and extractedwith ethyl acetate. After the organic layer was dried, the solvent wasevaporated under reduced pressure. The residue was purified by silicagel column chromatography to obtain the title compound (456 mg).

¹H-NMR (300MHz, CDCl₃) δ: 1.35 (d, 6H), 1.41 (t, 3H), 1.53 (t, 3H), 3.15(m, 1H), 4.39 (q, 2H), 4.52 (q, 2H), 6.94 (s, 1H), 7.44 (d, J=15.8Hz,1H), 7.47 (d, 1H), 7.94 (d, J=15.8Hz, 1H), 8.63 (s, 1H), 8.73 (d, 1H)

(B)1-Ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro[1,8]-naphthylidine-3-carboxylicacid

The ethyl1-ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro[1,8]naphthylidine-3-carboxylate(120 mg) obtained in (A) and lithium hydroxide (26 mg) were added withmethanol (10 ml) and water (5 ml), and then the mixture was stirred atroom temperature for 16 hours. After the methanol was evaporated, pH ofthe residue was made 6 by using 5% hydrochloric acid, and insolublesubstance was collected by filtration and dried to obtain the titlecompound (80 mg).

¹H-NMR (300MHz, CDCl₃) δ: 1.39 (d, 6H), 1.61 (t, 3H), 3.25 (m, 1H), 4.69(q, 2H), 7.05 (s, 1H), 7.62 (m, 2H), 8.13 (d, J=16Hz, 1H), 8.80 (d,J=8Hz, 1H), 8.94 (s, 1H)

MS (ES+) m/z 370 (M⁺+1), MS (ES−) m/z 368 (M⁺−1)

Example 1551-Ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-3-(1H-1,2,3,4-tetrazol-5-yl)-1,4-dihydro[1,8]naphthylidin-4-one

(A)1-Ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro[1,8]-naphthylidine-3-carboxamide

Eethyl1-ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro[1,8]naphthylidine-3-carboxylate(160 mg) was added with 25% aqueous ammonia (20 ml) and isopropanol (3ml) and heated at 100° C. for 16 hours in a Parr acid digestion bomb.After the reaction mixture was cooled, the solvent was evaporated toobtain the title compound (148 mg).

¹H-NMR (300MHz, CDCl₃) δ: 1.36 (d, 6H), 1.56 (t, 3H), 3.18 (m, 1H), 4.61(q, 2H), 5.75 (brs, 1H) 6.97 (s, 1H), 7.47 (d, J=15.8Hz, 1H), 7.51 (d,1H), 7.98 (d, J=15.8Hz, 1H), 8.75 (d, 1H), 8.95 (s, 1H), 9.56 (brs, 1H)

(B)1-Ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro[1,8]-naphthylidine-3-carbonitrile

The1-ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro[1,8]naphthylidine-3-carboxamide(32 mg) obtained in (A) was dissolved in 1,2-dichloroethane, added withbenzenesulfonyl chloride (0.12 ml), pyridine (0.18 ml) anddimethylaminopyridine (several pieces), and then the mixture was stirredat 40° C. for 24 hours. The reaction mixture was diluted with1,2-dichloroethane, then washed with 5% hydrochloric acid, saturatedaqueous sodium hydrogencarbonate and saturated brine, and then dried andthe solvent was evaporated under reduced pressure. The residue waspurified by silica gel column chromatography to obtain the titlecompound (19 mg).

¹H-NMR (300MHz, CDCl₃) δ: 1.36 (d, 6H), 1.56 (t, 3H), 3.18 (m, 1H), 4.56(q, 2H), 6.99 (s, 1H), 7.45 (d, J=15.8Hz, 1H), 7.51 (d, 1H), 7.98 (d,J=15.8Hz, 1H), 8.20 (s, 1H), 8.69 (d, 11H)

(C)1-Ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-3-(1H-1,2,3,4-tetrazol-5-yl)-1,4-dihydro[1,8]naphthylidin-4-one

The1-ethyl-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro[1,8]naphthylidine-3-carbonitrile(32 mg) obtained in (B) was dissolved in tetrahydrofuran (20 ml), addedwith sodium azide (100 mg), ammonium chloride (200 mg) and stirred at80° C. for 2 hours. The reaction mixture was added with water andadjusted to pH 8 with saturated aqueous sodium hydrogencarbonate, andthe tetrahydrofuran was evaporated. The residue was added with fivedrops of 25% aqueous ammonia, and insoluble solids were removed byfiltration. The filtrate was adjusted to pH 7 with 12% aqueoushydrochloric acid, loaded on a HP-20 reverse phase column, sufficientlywashed with water, and then eluted with water/acetonitrile/aqueousammonia (80:20:0.2, v/v). After the solvent was evaporated, the residuewas added with tert-butyl methyl ketone, and insoluble substance wastaken by filtration and dried to obtain the title compound (13 mg) asyellow powder.

¹H-NMR (300MHz, CDCl₃+CD₃OD) δ: 1.24 (d, 6H), 1.50 (t, 3H), 3.02 (m,1H), 4.61 (q, 2H), 6.92 (s, 1H), 7.40 (d, J=15.8Hz, 1H), 7.51 (d, J=8Hz,1H), 7.91 (d, J=15.8Hz, 1H), 8.66 (d, J=8Hz, 1H), 9.04 (s, 1H)

MS (ES+) m/z 394 (M⁺+1), MS (ES−) m/z 392 (M⁺−1)

Example 156(E)-3-{7-Fluoro-2-(3-hydroxypiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) N¹-(4-Methyl-5-nitro-2-pyridyl)acetamide

4-Methyl-5-nitro-2-pyridinamine (3.69 g, 2.41 mmol) was added withacetic anhydride (10 ml) and stirred at 130° C. for 1.5 hours. Thereaction mixture was left stand for cooling, and then added withdistilled water (5.4 ml) at 0° C., heated to 130° C., and stirred for 45minutes. The reaction mixture was left stand for cooling andconcentrated, and the deposited crystals were collected by filtrationand washed with distilled water to obtain the title compound (4.71 g,quantitative).

¹H-NMR (CDCl₃) δ: 2.26 (3H, s), 2.70 (3H, s), 8.19 (1H, br), 8.23 (1H,s), 8.95 (1H, s)

MS; m/z: MH 194

(B) N¹-(5-Amino-4-methyl-2-pyridyl)acetamide

The N1-(4-methyl-5-nitro-2-pyridyl)acetamide (4.70 g, 24.1 mmol)obtained in

(A) was dissolved in ethanol (150 ml), added with 10% palladium/carbon(0.95 g) and subjected to catalytic reduction overnight at 1 atm underhydrogen atmosphere. The catalyst was removed by filtration and washedwith ethanol. The filtrate was concentrated and the resulting residuewas purified by silica gel column chromatography(chloroform→chloroform:methanol=100:5) to obtain the title compound(4.10 g, quantitative) as brown solid.

¹H-NMR (CD₃OD) δ: 2.11 (3H, s), 2.18 (3H, s), 7.61 (1H, s), 7.70 (1H, s)

(C) 6-(Acetylamino)-4- methyl-3-pyridinediazonium tetrafluoroborate

The N¹-(5-amino-4-methyl-2-pyridyl)acetamide (8.00 g, 48.4 mmol)obtained in (B) was dissolved in tetrafluoroboric acid (160 ml) andslowly added dropwise with an aqueous solution (40 ml) of sodium nitrite(3.51 g, 50.8 mmol) at −20° C. under nitrogen atmosphere. The reactionmixture was further stirred at −10° C. for 1 hour and added with diethylether (800 ml), and the deposited white solid was collected byfiltration and washed with diethyl ether to obtain the title compound(14.7 g, quantitative).

(D) N¹-(5-Fluoro-4-methyl-2-pyridyl)acetamide

Toluene (280 ml) heated at 100° C. with stirring was added with the6-(acetylamino)-4-methyl-3-pyridinediazonium tetrafluoroborate (12.8 g,48.4 mmol) obtained in (C) and further refluxed by heating for 1 hour.After the reaction mixture was left stand for cooling and the solventwas evaporated, the residue was diluted with chloroform and washed with1 N aqueous sodium hydroxide. The organic layer was dried over anhydrousmagnesium sulfate, then the solvent was evaporated and the resultingresidue was purified by silica gel column chromatography (chloroform:methanol=100:1) to obtain the title compound (3.48 g, 43%) as yellowsolid.

¹H-NMR (CDCl₃) δ: 2.19 (3H, s), 2.32 (3H, s), 7.93 (1H, br), 7.98 (1H,s), 8.08 (1H, d, J=5.6Hz)

MS; m/z: (MH⁺) 169

(E) 5-Fluoro-4-methyl-2-pyridinamine

The N¹-(5-fluoro-4-methyl-2-pyridyl)acetamide (4.25 g, 25.3 mmol)obtained in (D) was dissolved in ethanol (3 ml), added with 6 M aqueoushydrochloric acid (3 ml) and refluxed by heating for 1 hour and 30minutes. The reaction mixture was left stand for cooling and the solventwas concentrated. The resulting residue was dissolved in distilledwater, and the system was made basic with 1 N aqueous sodium hydroxideand extracted twice with chloroform. The organic layer was dried overanhydrous magnesium sulfate and the solvent was evaporated to obtain thetitle compound (2.82 g, 88%) as solid.

¹H-NMR (CD₃OD) δ: 2.19 (3H, s), 6.45 (1H, m), 7.65 (1H, m)

ES-MS; m/z: (MH⁺) 127

(F) tert-Butyl N-(5-fluoro-4-methyl-2-pyridyl)carbamate

The 5-fluoro-4-methyl-2-pyridinamine (2.82 g, 22.4 mmol) obtained in (E)was dissolved in tert-butanol (100 ml), slowly added dropwise with asolution of di-tert-butyl dicarbonate (5.12 g, 23.5 mmol) intetrahydrofuran (10 ml) over 1 hour, and then the mixture was stirred atroom temperature. The deposited substance was removed by filtration, andthe reaction mixture was concentrated. The resulting residue waspurified by silica gel column chromatography (chloroform) to obtain thetitle compound (3.17 g, 66%) as white solid.

¹H-NMR (CDCl₃) δ: 1.53 (9H, s), 2.30 (3H, s), 7.74 (1H, br), 7.83 (1H,d, J=5.8Hz), 7.99 (1H, d, J=1.2Hz)

MS; m/z: (MH⁺) 227

(G) tert-ButylN-{5-fluoro-4-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-pyridyl}carbamate

The tert-butyl N-(5-fluoro-4-methyl-2-pyridyl)carbamate (75.6 mg, 0.334mmol) obtained in (F) was dissolved in tetrahydrofuran (2 ml) and addeddropwise with n-butyl lithium (1.5 M solution in hexane, 0.468 ml, 0.702mmol) at −78° C. under argon atmosphere. The reaction mixture was warmedto room temperature, stirred for 1 hour, cooled to −78° C. again, addeddropwise with a solution of 2-(bromomethyl)-4-isopropyl-1,3-thiazole(84.6 mg, 0.384 mmol) in tetrahydrofuran (2 ml), and then warmed to roomtemperature. The reaction mixture was added with water and extractedtwice with ethyl acetate. The organic layer was dried over anhydrousmagnesium sulfate, and the solvent was evaporated. The resulting residuewas purified by silica gel column chromatography (chloroform:ethylacetate=100:1) to obtain the title compound (40.0 mg, 33%).

¹H-NMR (CDCl₃) δ: 1.28 (6H, d, J=7.1Hz), 1.54 (9H, s), 3.09 (1H, m),3.15 (2H, m), 3.32 (2H, m), 6.71 (1H, s), 7.90 (1H, d, J=5.6Hz), 8.05(1H, d, J=1.5Hz)8.23 (1H, br)

MS; m/z: (MH⁺) 366

(H) 5-Fluoro-4-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-pyridinamine

The tert-butylN-{5-fluoro-4-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-pyridyl}-carbamate(393 mg, 1.08 mmol) obtained in (G) was dissolved in methylene chloride(10 ml), added with trifluoroacetic acid (10 ml) under ice cooling, andstirred overnight at room temperature. The reaction mixture wasconcentrated, and the resulting residue was dissolved in chloroform andwashed with 1 N aqueous sodium hydroxide. The organic layer was driedover anhydrous magnesium sulfate and the solvent was evaporated. Theresulting residue was purified by silica gel column chromatography(chloroform:methanol=100:2) to obtain the title compound (40.0 mg, 33%)as an oil.

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.8Hz), 3.05 (3H, m), 3.27 (2H, m),6.32 (1H, m), 6.72 (1H, m), 7.84 (1H, m)

(I)7-Fluoro-2-hydroxy-8-1-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-4-one

The 5-fluoro-4-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-2-pyridinamine(310 mg, 1.15 mmol) obtained in (H) was dissolved in xylene (2.5 ml),added with di(2,4,6-trichlorophenyl)malonate (568 mg, 1.23 mmol), andrefluxed by heating for 30 minutes. The reaction mixture was left standfor cooling and then concentrated, and the resulting residue waspurified by silica gel column chromatography (chloroform:methanol=100:1)to obtain the title compound (325 mg, 84%) as yellow solid.

¹H-NMR (CDCl₃) δ: 1.27 (6H, d, J=6.8Hz), 3.05 (1H, m), 3.40 (4H, m),5.40 (1H, s), 6.75 (1H, s), 7.40 (1H, d, J=5.9Hz), 9.00 (1H, d, J=4.6Hz)

MS; m/z: (MH⁺) 334, (MH⁻) 332

(J)7-Fluoro-2-(3-hydroxypiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-4-one

The7-fluoro-2-hydroxy-8-(2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-4-one(150 mg, 0.450 mmol) obtained in (I) was dissolved in a mixed solvent oftetrahydrofuran (3 ml) and dimethylformamide (1 ml), added withp-toluenesulfonyl chloride (129 mg, 0.675 mmol) and4-dimethylaminopyridine (60.5 mg, 0.494 mmol), and then the mixture wasstirred at room temperature for 2 hours.

The reaction mixture was diluted with chloroform, washed with water anddried over anhydrous magnesium sulfate. The solvent was evaporated andthe resulting residue was dissolved in dimethylformamide (3 ml), addedwith 3-hydroxypiperidine (310 mg, 2.25 mmol) and triethylamine (0.3 ml)and stirred at 80° C. for 2 hours. The reaction mixture was left standfor cooling, diluted with chloroform, washed with water and dried overanhydrous magnesium sulfate. Then, the solvent was evaporated and theresulting residue was purified by preparative TLC(chloroform:methanol=100:3) to obtain the title compound (147 mg, 79%)as yellow solid.

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.8Hz), 1.56 (1H, m), 1.64 (1H, m),1.86 (1H, m), 1.99 (1H, m), 3.07 (1H, m), 3.21 (2H, m), 3.34 (1H, m),3.36 (2H, m), 3.38 (1H, m), 3.82 (1H, m), 3.98 (1H, m), 5.66 (1H, s),6.73 (1H, s), 7.11 (1H, d, J=6.6Hz), 8.70 (1H, d, J=5.4Hz)

(K)1-{7-Fluoro-3-formyl-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}-3-piperidylformate

Dimethylformamide (3 ml) was added with phosphorus oxychloride (0.083ml, 0.882 mmol) under ice cooling and stirred for 30 minutes. Themixture was added with a solution of the7-fluoro-2-(3-hydroxypiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-4-one(147 mg, 0.353 mmol) obtained in (J) in dimethylformamide (2 ml), warmedto room temperature, and then stirred for 1 hour. The reaction mixturewas further added with phosphorus oxychloride (0.083 ml, 0.882 mmol) andstirred for 30 minutes. The reaction mixture was added with saturatedaqueous sodium hydrogencarbonate and extracted twice with ethyl acetate.The organic layer was dried over anhydrous magnesium sulfate, and thesolvent was evaporated. The resulting residue was purified by silica gelcolumn chromatography (chloroform→chloroform:methanol=400:1) and furtherpurified by preparative TLC (chloroform:methanol=100:1) to obtain thetitle compound (145 mg, 87%) as yellow oil.

¹H-NMR (CDCl₃) δ: 1.28 (6H, d, J=6.8Hz), 1.69 (1H, m), 1.85 (1H, m),1.89 (1H, m), 2.02 (1H, m), 3.06 (1H, m), 3.25 (2H, m), 3.37 (2H, m),3.63 (2H, m), 3.78 (1H, dd, J=13.7 and 6.6Hz), 3.88 (1H, dd, J=13.7 and3.2Hz), 5.06 (1H, m), 6.74 (1H, s), 7.10 (1H, d, J=6.6Hz), 7.98 (1H, s),8.69 (1H, d, J=5.4Hz), 10.1 (1H, s)

(L) tert-Butyl(E)-3-{7-fluoro-2-(3-formylpiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)-ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

The1-{7-fluoro-3-formyl-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}-3-piperidylformate (145 mg, 0.307 mmol) obtained in (K) was dissolved intetrahydrofuran (5 ml), added withtert-butoxycarbonylmethylenetriphenylphosphorane (1.38 g, 3.68 mmol) andrefluxed by heating for 2 days. The reaction mixture was concentrated,and the resulting residue was purified by preparative TLC(chloroform:methanol=100:4) to obtain the title compound (147 mg, 84%)as yellow solid.

¹H-NMR (CDCl₃) δ: 1.28 (6H, d, J=6.9Hz), 1.51 (9H, s), 1.73 (1H, m),1.85 (1H, m), 1.99 (2H, m), 3.06 (1H, m), 3.26 (2H, m), 3.38 (2H, m),3.48 (1H, m), 3.52 (1H, m), 3.65 (1H, m), 3.74 (1H, m), 5.12 (1H, m),6.73 (1H, s), 7.05 (1H, d, J=15.5Hz), 7.21 (1H, d, J=6.9Hz), 7.48 (1H,d, J=15.5Hz), 8.07 (1H, s), 8.80 (1H, d, J=5.4Hz)

(M) tert-Butyl(E)-3-{7-fluoro-2-(3-hydroxypiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

The tert-butyl(E)-3-{7-fluoro-2-(3-formylpiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(147 mg, 0.258 mmol) obtained in (L) was dissolved in methanol (3 ml),added with sodium methoxide (33.6 mg, 0.618 mmol), and then stirred atroom temperature for 9 hours. The reaction mixture was added withdistilled water and extracted with chloroform (twice). The organic layerwas dried over anhydrous magnesium sulfate, and the solvent wasevaporated. The resulting residue was purified by preparative TLC(chloroform:methanol=100:5) to obtain the title compound (91.6 mg, 65%).

¹H-NMR (CDCl₃) δ: 1.27 (6H, d, J=6.8Hz), 1-51 (9H, s), 1.60 (2H, m),1.83 (2H, m), 3.05 (1H, m), 3.26 (2H, m), 3.38 (2H, m), 3.53 (1H, m),3.57 (2H, m), 3.73 (1H, m), 3.87 (1H, m), 4.01 (1H, m), 6.73 (1H, s),7.03 (1H, d, J=15.6Hz), 7.21 (1H, d, J=6.6Hz), 7.48 (1H, d, J=15.6Hz),8.81 (1H, d, J=5.6Hz)

(N) (E)3-{7-Fluoro-2-(3-hydroxypiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

The tert-butyl(E)-3-{7-fluoro-2-(3-hydroxypiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(43.6 mg, 0.0803 mmol) obtained in (M) was dissolved in 4 N hydrochloricacid/dioxane (3 ml) and stirred at room temperature for 2 hours. Thereaction mixture was concentrated and the resulting residue was purifiedby preparative TLC (chloroform:methanol=10:1) to obtain the titlecompound (35.6 mg, 91%) as lyophilized product.

¹H-NMR (CDCl₃) δ: 1.27 (6H, d, J=6.8Hz), 1.54 (1H, m), 1.71 (1H, m),1.87 (2H, m), 3.06 (1H, m), 3.23 (2H, m), 3.37 (2H, m), 3.45 (1H, m),3.62 (3H, m), 3.99 (1H, m), 6.73 (1H, s), 7.00 (1H, d, J=15.4Hz), 7.18(1H, d, J=6.6Hz), 7.56 (1H, d, J=15.4Hz), 8.73 (1H, d, J=4.9Hz)

MS; m/z: (MH⁺) 487, (MH⁻) 485

Example 157(E)-3-{2-{3-[(Aminocarbonyl)oxy]piperidino}-7-fluoro-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl (E)3-{2-{3-[(aminocarbonyl)oxy]piperidino}-7-fluoro-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

The tert-butyl(E)-3-{7-fluoro-2-(3-hydroxypiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(48.0 mg, 0.0885 mmol) obtained in Example 156, (M) was dissolved inethyl acetate (3 ml), added with trichloroacetyl isocyanate (0.052 ml,0.442 mmol) and stirred at room temperature for 1 hour. The reactionmixture was added with methanol:chloroform (1:10, 10 ml) andconcentrated, and the resulting residue was dissolved in a mixed solventof methanol (3 ml) and distilled water (1 ml), added with sodium formate(12.0 mg, 0.177 mmol) and stirred overnight at room temperature. Thesolution was further added with sodium formate (12.0 mg, 0.177 mmol),stirred for 5 hours and then concentrated, and the resulting residue waspurified by preparative TLC (chloroform:methanol=100:5) to obtain thetitle compound (71.3 mg, quantitative) as lyophilized product.

¹H-NMR (CDCl₃) δ: 1.27 (6H, d, J=6.8Hz), 1 51 (9H, s), 1.78 (1H, m),1.84-2.00 (3H, m), 3.06 (1H, m), 3.26 (2H, m), 3.27 (1H, m), 3.39 (2H,m), 3.52 (1H, m), 3.66.(2H, m), 4.81 (1H, m), 4.90 (1H,br), 6.70(1H,br), 6.74 (1H, s), 7.08 (1H, d, J=15.6Hz), 7.26 (1H, d, J=6.8Hz),7.68 (1H, d, J=15.6Hz), 8.81 (1H, d, J=5.4Hz)

MS; m/z: (MH⁺) 586, (MH⁻) 584

(B)(E)-3-{2-{3-[(Aminocarbonyl)oxy]piperidino}-7-fluoro-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

The tert-butyl(E)-3-{2-{3-[(aminocarbonyl)oxy]piperidino}-7-fluoro-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(51.8 mg, 0.0884 mmol) obtained in (A) was dissolved in 4 N hydrochloricacid in dioxane (3 ml) and stirred at room temperature. The reactionmixture was concentrated and the resulting residue was purified bypreparative TLC (chloroform:methanol=10:1) to obtain the title compound(26.8 mg, 57%) as lyophilized product.

¹H-NMR (CD₃OD) δ: 1.25 (6H, d, J=6.8Hz), 1.68 (1H, m), 1.82 (1H, m),1.97 (2H, m), 3.02 (1H, m), 3.25 (2H, m), 3.41 (2H, m), 3.48 (1H, m),3.57 (1H, m), 3.66 (1H, m), 3.77 (1H, m), 4.73 (1H, m), 6.94 (1H, d,J=15.6Hz), 6.97 (1H, s), 7.29 (1H, d, J=6.8Hz), 7.55 (1H, d, J=15.6Hz),8.71 (1H, d, J=5.6Hz)

MS; m/z: (MH⁺) 530, (MH⁻) 528

Example 1583-{2-(3-Hydroxypiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}propanoicacid

(A) Methyl(E)-3-{2-(3-formylpiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

1-{3-Formyl-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}-3-piperidylformate(94.0 mg, 0.207 mmol) andbis(2,2,2-trifluoroethyl)(methoxycarbonylmethyl)phosphonoate (0.131 ml,0.620 mmol) were dissolved in tetrahydrofuran (2 ml), added with DBU(0.085 ml, 0.620 mmol) and lithium chloride (26.3 mg, 0.620 mmol), andthen the mixture was stirred at room temperature for 1 hour.

The reaction mixture was concentrated and the obtained residue waspurified by preparative TLC (chloroform:methanol=100:2) to obtain thetitle compound (80.9 mg, 76%).

¹H-NMR (CDCl₃) δ: 1.28 (6H, d, J=6.8Hz), 1.74-2.00 (4H, m), 3.07 (1H,m), 3.20 (2H, t, J=7.8Hz), 3.36 (2H, t, J=7.8Hz), 3.54 (2H, m), 3.67(1H, m), 3.76 (1H, m), 3.77 (3H, s), 5.14 (1H, m), 6.73 (1H, s), 6.84(1H, dd, J=7.3 and 1.7Hz), 7.10 (1H, d, J=15.6Hz), 7.20 (1H, s), 7.62(1H, d, J=15.6Hz), 8.09 (1H, s), 8.85 (1H, d, J=7.3Hz)

(B) Methyl3-{2-(3-formylpiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}propanoate

The methyl(E)-3-{2-(3-formylpiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(80.9 mg, 0.158 mmol) obtained in (A) was dissolved in ethanol (3 ml),added with 5% palladium/carbon (30 mg) and stirred at 1 atm for 2 daysunder hydrogen atmosphere. The catalyst was removed by filtration, andthe filtrate was concentrated. The resulting residue was purified bypreparative TLC (chloroform:methanol=100:2) to obtain the title compound(23.1 mg, 28%).

¹H-NMR (CDCl₃) δ: 1.29 (6H, d, J=6.8Hz), 1.74-2.03 (4H, m), 3.07 (1H,m), 3.20 (2H, t, J=7.8Hz), 2.73 (2H, m), 2.93 (2H, m), 3.15 (1H, m),3.20 (2H, m), 3.30 (2H, m), 3.35 (2H, m), 3.37 (2H, m), 3.68 (3H, s),5.12 (1H, m), 6.72 (1H, s), 6.81 (1H, d, J=7.3Hz), 7.20 (1H, s), 7.26(1H, s), 8.07 (1H, s), 8.79 (1H, d, J=7.3Hz)

MS; m/z: 513 (MH⁺), 511 (MH⁻)

(C)3-{2-(3-Hydroxypiperidino)-8-[2.-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}propanoicacid

The methyl3-{2-(3-formylpiperidino)-8-[2-(4-isopropyl-1,3-thiazol-2-yl)ethyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]propanoate(23.1 mg, 0.0451 mmol) obtained in (B) was dissolved in a mixed solutionof methanol, tetrahydrofuran and water (1:1:1, 3 ml), added with lithiumhydroxide monohydrate (3.8 mg, 0.0901 mmol), and stirred overnight atroom temperature. The reaction mixture was added with 1 N hydrochloricacid (0.091 ml) to neutralize the system, and then the solvent wasevaporated. The resulting residue was purified by preparative TLC(chloroform:methanol=10:1) to obtain the title compound (15.0 mg, 71%)as lyophilized product.

¹H-NMR (CD₃OD) δ: 1.26 (6H, d, J=6.8Hz), 1.51 (1H, m), 1.67 (1H, m),1.84 (1H, m), 1.99 (1H, m), 2.64 (2H, m), 2.90 (2H, m), 2.95-3.08 (3H,m), 3.20 (2H, t, J=7.3Hz), 3.40 (2H, t, J=7.3Hz), 3.57 (1H, m), 3.78(2H, m), 6.96 (1H,s), 7.03 (1H, dd, J=7.3 and 2.0Hz), 7.21 (1H, s), 8.74(1H,d, J=7.3Hz)

MS; m/z: 471 (MH⁺), 469 (MH⁻)

Example 1591-(2-Fluoroethyl)-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-3-(1H-1,2,3,4-tetrazol-5-yl)-1,4-dihydro-4-quinolinone

(A) Ethyl1-(2-fluoroethyl)-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro-3-quinolinecarboxylate

Ethyl7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl)-4-oxo-1,4-dihydro-3-quinolinecarboxylate(368 mg, 1.00 mmol) was dissolved in dimethylformamide (6 ml), addedwith potassium carbonate (276 mg, 2.00 mmol) and 1-bromo-2-fluoroethane(0.223 ml, 3.00 mmol), and then the mixture was stirred overnight at 65°C. The reaction mixture was left stand for cooling and then diluted withethyl acetate. The organic layer was washed with water and dried overanhydrous magnesium sulfate, and the solvent was evaporated. Theresulting residue was purified by silica gel column chromatography(chloroform→chloroform:methanol=100:1) to obtain the title compound (254mg, 61%) as colorless transparent oil.

¹H-NMR (CDCl₃) δ: 1.34 (6H, d, J=7.1Hz), 1.39 (3H, t, J=7.1Hz), 3.13(1H, m), 4.36 (2H,q), 4.54 (2H, ddd, J=24.9, 4.6 and 4.4Hz), 4.85 (2H,ddd, J=46.6, 4.6 and 4.4Hz), 6.86 (1H, s), 7.40 (2H, s), 7.43 (1H, s),7.57 (1H, dd, J=8.5 and 1.2Hz), 8.43 (1H, s), 8.47 (1H, dd, J=8.5 and2.0Hz)

MS; m/z: (MH⁺) 415

(B)1-(2-Fluoroethyl)-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro-3-quinolinecarboxylicacid

The ethyl1-(2-fluoroethyl)-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro-3-quinolinecarboxylate(254 mg, 0.613 mmol) obtained in (A) was dissolved in a mixed solutionof methanol, tetrahydrofuran and water (1:1:1), added with 1 N aqueoussodium hydroxide (1.23 ml, 1.23 mmol), and then the mixture was stirredat room temperature for 8 hours. The reaction mixture was added with 1 Nhydrochloric acid (1.23 ml, 1.23 mmol) to neutralize the system, andextracted with chloroform. The organic layer was dried over anhydrousmagnesium sulfate, and the solvent was evaporated to obtain the titlecompound (174 mg, 73%) as yellow solid.

(C)1-(2-Fluoroethyl)-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro-3-quinolinecarboxamide

The1-(2-fluoroethyl)-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro-3-quinolinecarboxylicacid (174 mg, 0.450 mmol) obtained in (B) was dissolved indimethylformamide (3 ml), added with triethylamine (0.126 ml, 0.900mmol) and ethyl chloroformate (0.086 ml, 0.900 mmol) under ice cooling,and then the mixture was stirred for 1 hour. The reaction mixture waswarmed to room temperature, stirred for 30 minutes and further stirredat 0° C. for 1 hour. The reaction mixture was added with concentratedaqueous ammonia (0.15 ml) and stirred overnight at room temperature. Thereaction mixture was diluted with ethyl acetate and washed successivelywith aqueous citric acid, saturated aqueous sodium hydrogencarbonate andsaturated brine and dried over anhydrous magnesium sulfate. Then, thesolvent was evaporated to obtain the title compound (164 mg, 94%) aspale yellow solid.

¹H-NMR (CDCl₃) δ: 1.36 (6H, d, J=6.8Hz), 3.17 (1H, m), 4.59 (2H, ddd,J=23.9, 4.9 and 4.6Hz), 4.87 (2H, ddd, J=46.4, 4.9 and 4.6Hz), 5.75(1H,br), 6.92 (1H, s), 7.28 (1H, m), 7.47 (2H, s), 7.69 (1H, d,J=8.6Hz), 8.54 (1H, d, J=8.6Hz), 8.79 (1H, s), 9.66 (1H, br)

MS; m/z: (MH⁺) 386

(D)1-(2-Fluoroethyl)-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro-3-quinolinecarbonitrile

A solution of dimethylformamide (0.109 ml, 1.40 mmol) in acetonitrile (2ml) was added with oxalyl chloride (0.111 ml, 1.28 mmol) under icecooling, stirred at the same temperature for 15 minutes, and then addedwith a solution of the1-(2-fluoroethyl)-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro-3-quinolinecarboxamide(164 mg, 0.425 mmol) obtained in (C) in acetonitrile (3 ml). The mixturewas stirred at the same temperature for 10 minutes. This mixture wasadded with pyridine (0.206 ml, 2.55 mmol) and stirred at the sametemperature for 10 minutes and further at room temperature for 2 hours.The reaction mixture was added with a solution of dimethylformamide(0.109 ml, 1.40 mmol) and oxalyl chloride (0.111 ml, 1.28 mmol) inacetonitrile (1 ml) prepared beforehand at 0° C., then added withpyridine (0.206 ml, 2.55 mmol), and stirred at room temperature for 1hour. The reaction mixture was added with ethyl acetate and washed withaqueous citric acid, saturated aqueous sodium hydrogencarbonate andsaturated brine. The organic layer was dried over anhydrous magnesiumsulfate, and the solvent was evaporated. The resulting residue waspurified by preparative TLC (chloroform:methanol=100:7) to obtain thetitle compound (179 mg, quantitative) as pale yellow as solid.

¹H-NMR (CDCl₃) δ: 1.36 (6H, d, J=7.1Hz), 3.15 (1H, m), 4.52 (2H, ddd,J=24.6, 4.6 and 4.4Hz), 4.85 (2H, ddd, J=46.6, 4.6 and 4.4Hz), 6.93 (1H,s), 7.43 (1H, s), 7.46 (2H, s), 7.69 (1H, dd, J=8.5 and 1.2Hz), 8.03(1H, s), 8.49 (1H, d, J=8.6Hz)

(E)1-(2-Fluoroethyl)-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-3-(1H-1,2,3,4-tetrazol-5-yl)-1,4-dihydro-4-quinolinone

A solution of aluminum chloride (55.7 mg, 0.418 mmol) indimethylformamide (1 ml) was added with sodium azide (81.5 mg, 1.25mmol) under ice cooling, and then stirred at room temperature for 15minutes. Then, the reaction mixture was added with a solution of the1-(2-fluoroethyl)-7-[(E)-2-(4-isopropyl-1,3-thiazol-2-yl)-1-ethenyl]-4-oxo-1,4-dihydro-3-quinolinecarbonitrile(51.2 mg, 0.139 mmol) obtained in (D) in dimethylformamide (1 ml) andstirred overnight at 85-90° C. The reaction mixture was poured into icewater/1N hydrochloric acid (1 ml) and stirred at room temperature for1.5 hours. The precipitates were collected by filtration andrecrystallized from chloroform/methanol/hexane to obtain the titlecompound (15.1 mg, 26%) as colorless solid.

¹H-NMR (DMSO-d₆) δ: 1.29 (6H, d, J=7.1Hz), 3.08 (1H, m), 4.82 (1H, m),4.94 (2H, m), 4.95 (1H, m), 7.34 (1H, s), 7.64 (1H, d, J=16.2Hz), 7.84(1H, d, J=16.2Hz), 7.96 (1H, d, J=8.6Hz), 8.23 (1H, s), 8.37 (1H, d,J=8.6Hz), 9.05 (1H, s)

LCMS; m/z: 411 (MH⁺)

Example 1601-Cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-2-morpholino-4-oxo-1,4-dihydro-3-quinolinecarboxylicacid

(A) Ethyl1-cyclopropyl-6,7-difluoro-2-(methylsulfonyl)-4-oxo-1,4-dihydro-3-quinolinecarboxylate

Ethyl1-cyclopropyl-6,7-difluoro-2-(methylsulfanyl)-4-oxo-1,4-dihydro-3-quinolinecarboxylate(300 mg, 0.976 mmol), which is a known compound [J. Heterocyclic Chem.,27, 839 (1990)], was dissolved in methylene chloride (6 ml), added withmetachloroperformic acid (755 mg, 2.93 mmol) and stirred overnight. Thereaction mixture was diluted with chloroform and washed successivelywith saturated aqueous sodium hydrogencarbonate and sodiumhydrogensulfite. The organic layer was dried over anhydrous sodiumsulfate, and the solvent was evaporated. The resulting residue wasdeveloped by silica gel column chromatography (hexane:ethyl acetate=2:1)to obtain the title compound (140 mg, 39%) as white solid. The resultingcompound was further developed (chloroform:methanol=100:1) to obtainethyl1-cyclopropyl-6,7-difluoro-2-(methylsulfinyl)-4-oxo-1,4-dihydro-3-quinolinecarboxylate(101 mg, 29%) as colorless transparent oil.

Sulfone Compound (Title Compound)

¹H-NMR (CDCl₃) δ: 0.88 (2H, m), 1.39 (3H, t, J=7.1Hz), 1.44 (2H, m),3.56 (3H, s), 3.92 (1H, m), 4.43 (2H,q, J=7.1Hz), 7.63 (1H, dd, J=11.5and 6.4Hz), 8.01 (1H, dd, J=9.8 and 8.6Hz)

ES-MS; m/z: 372 (MH⁺)

Sulfoxide Compound

¹H-NMR (CDCl₃) δ: 0.99 (2H, m), 1.39 (3H, t, J=6.9Hz), 1.46 (2H, m),3.16 (3H, s), 4.42 (3H, m), 7.66 (1H, dd, J=11.3 and 6.4Hz), 8.13 (1H,t, J=9.3Hz)

ES-MS; m/z: 356 (MH⁺)

(B) Ethyl1-cyclopropyl-6,7-difluoro-2-morpholino-4-oxo-1,4-dihydro-3-quinolinecarboxylate

The ethyl1-cyclopropyl-6,7-difluoro-2-(methylsulfonyl)-4-oxo-1,4-dihydro-3-quinolinecarboxylate(140 mg, 0.377 mmol) obtained in (A) was dissolved in tetrahydrofuran (5ml), added with morpholine (0.0395 ml, 0.452 mmol),N,N-diisopropylethylamine (0.131 ml, 0.754 mmol) and magnesium bromidediethyl etherate (389 mg, 1.51 mmol), and then the mixture was refluxedby heating for 2 hours. The reaction mixture was diluted with ethylacetate and washed with water. The organic layer was dried overmagnesium sulfate, and the solvent was evaporated under reducedpressure. The resulting residue was purified by preparative TLC(chloroform:methanol=100:5) to obtain the title compound (89.2 mg, 62%).

¹H-NMR (CDCl₃) δ: 0.80 (2H, m), 1.40 (3H, t, J=7.1Hz), 1.41 (2H, m),3.14 (1H, m), 3.36 (4H, t, J=4.4Hz), 3.84 (4H, t, J=4.4Hz), 4.38 (2H,q,J=7.1Hz), 7.47 (1H, dd, J=11.7 and 6.4Hz), 8.01 (1H, dd, J=10.0 and9.0Hz)

(C) Ethyl1-cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-2-morpholino-4-oxo-1,4-dihydro-3-quinolinecarboxylate

(4-Isopropyl-1,3-thiazol-2-yl)methanol (37.0 mg, 0.235 mmol) wasdissolved in dimethylformamide (2 ml), added with 18-crown-6 (68.4 mg,0.259 mmol) and sodium hydride (95%, 6.5 mg, 0.259 mmol), and stirredfor 10 minutes under argon atmosphere. The reaction mixture was addedwith a solution of the ethyl1-cyclopropyl-6,7-difluoro-2-morpholino-4-oxo-1,4-dihydro-3-quinolinecarboxylate(89.0 mg, 0.235 mmol) obtained in (B) in dimethylformamide (1 ml), andstirred at room temperature for 1 hour. Then, the reaction mixture wasadded with aqueous ammonium chloride and extracted with ethyl acetate.The organic layer was dried over magnesium sulfate, and the solvent wasevaporated under reduced pressure. The resulting residue was purified bypreparative TLC (chloroform:methanol=100:5, v/v) to obtain the titlecompound (102 mg, 84%).

¹H-NMR (CDCl₃) δ: 0.68 (2H,m), 1.29 (2H, m), 1.32 (6H, d, J=6.8Hz), 1.39(3H, t, J=7.1Hz), 3.05 (1H, m), 3.11 (1H, m), 3.32 (4H, t, J=4.4Hz),3.82 (4H, t, J=4.4Hz), 4.39 (2H,q, J=7.1Hz), 5.52 (2H, s), 6.97 (1H, s),7.27 (1H, d, J=7.3Hz), 7.93 (1H, d, J=11.2Hz)

ES-MS; m/z: 516 (MH⁺)

(D)1-Cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-2-morpholino-4-oxo-1,4-dihydro-3-quinolinecarboxylicacid

A solution of aluminum chloride (266 mg, 2.00 mmol) in1,2-dichloroethane (4 ml) was added with dimethyl sulfide (0.293 ml,4.00 mmol) and stirred at 0° C. for 30 minutes. Then, the reactionmixture was added with ethyl1-cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-2-morpholino-4-oxo-1,4-dihydro-3-quinolinecarboxylate(51.5 mg, 0.0999 mmol) and refluxed overnight by heating. The reactionmixture was left stand for cooling, diluted with chloroform, washed with1 N hydrochloric acid, and dried over anhydrous magnesium sulfate. Thesolvent was evaporated and the obtained residue was purified bypreparative TLC (chloroform:methanol=10:1) to obtain the title compound(1.3 mg, 3%).

¹H-NMR (CDCl₃) δ: 0.58 (2H, m), 1.17 (2H, m), 1.33 (6H, d, J=6.8Hz),3.11 (1H, m), 3.29 (1H, m), 3.52 (4H, m), 4.00 (4H, m), 5.55 (2H, s),6.97 (1H, s), 7.28 (1H, d, J=6.8Hz), 7.90 (1H, d, J=10.7Hz)

LCMS; m/z: 487 (M)

Example 161(Z)-3-(1-Cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-4-oxo-1,4-dihydro-3-quinolyl)-2-propeonicacid

(A) 1-Cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydro-3- quinolinecarboxamide1-Cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid(1.00 g, 3.77 mmol) was dissolved in dimethylformamide (15 ml), addedwith triethylamine (0.788 ml, 5.66 mmol) and ethyl chloroformate (0.538ml, 5.66 mmol) under ice cooling, and stirred for 1 hour. The reactionmixture was warmed to room temperature, stirred for 30 minutes, andfurther stirred at 0° C. for 1 hour. The reaction mixture was added withconcentrated aqueous ammonia (0.75 ml) and stirred overnight at roomtemperature. The reaction mixture was diluted with ethyl acetate andwashed successively with aqueous citric acid, saturated aqueous sodiumhydrogencarbonate and saturated brine. The organic layer was dried overanhydrous magnesium sulfate, and the solvent was evaporated to obtainthe title compound (1.23 g, quantitative) as white solid.

¹H-NMR (CD₃OD) δ: 1.21 (2H, m), 1.42 (2H, m), 3.56 (1H, m), 7.88 (1H,dd, J=11.2 and 6.4Hz), 8.25 (1H, dd, J=10.5 and 8.5Hz), 8.88 (1H, s)

(B) 1-Cyclopropyl-6,7difluoro-4-oxo-1,4-dihydro3-quinolinecarbonitrile

A solution of dimethylformamide (0.712 ml, 9.19 mmol) in acetonitrile(10 ml) was added with oxalyl chloride (0.729 ml, 8.36 mmol) under icecooling, stirred at the same temperature for 15 minutes, added with asolution of the1-cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydro-3-quinolinecarboxamide(1.23 g, 4.18 mmol) obtained in (A) in acetonitrile (10 ml) and stirredat the same temperature for 10 minutes. This mixture was added withpyridine (1.35 ml, 16.7 mmol), stirred at the same temperature for 10minutes, and then stirred overnight at room temperature. The reactionmixture was added with ethyl acetate, washed successively with aqueouscitric acid, saturated aqueous sodium hydrogencarbonate and saturatedbrine, and dried over anhydrous magnesium sulfate to obtain the titlecompound (714 mg, 77%) as pale yellow solid.

¹H-NMR (CDCl₃) δ: 1.16 (2H, m), 1.42 (2H, m), 3.47 (1H, m), 7.78 (1H,dd, J=11.0 and 6.4Hz), 8.16 (1H, s), 8.21 (1H, dd, J=10.0 and 8.6Hz)

MS; m/z: 247 (MH⁺)

(C) 1-Cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydro-3-quinolinecarbaldehyde

The 1-cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydro-3-quinolinecarbonitrile(150 mg, 0.609 mmol) obtained in (B) was dissolved in a mixed solutionof acetic acid, water and pyridine (1:1:2, 4 ml), added with RaneyNickel (catalytic amount) and sodium phosphinate monohydrate (258 mg,2.44 mmol), and then the mixture was stirred overnight at 60° C. Thereaction mixture was left stand for cooling and the catalyst was removedby filtration through a Celite layer and washed with hot ethanol. Thereaction mixture was concentrated, then diluted with chloroform, andwashed with aqueous copper sulfate. The organic layer was dried overanhydrous magnesium sulfate to obtain the title compound (97.0 mg, 64%)as pale yellow solid.

¹H-NMR (CDCl₃) δ: 1.18 (2H, m), 1.39 (2H, m), 3.47 (1H, m), 7.78 (1H,dd, J=11.2 and 6.3Hz), 8.27 (1H, dd, J=10.2 and 8.8Hz), 8.42 (1H, s),10.37 (1H, s)

LCMS; m/z: 250 (MH⁺)

(D) tert-Butyl (E)- and(Z)-3-(1-cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydro-3-quinolyl)-2-propenoate

The 1-cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydro-3-quinolinecarbaldehyde(97.0 mg, 0.389 mmol) obtained in (C) was dissolved in a mixed solventof dimethylformamide and tetrahydrofuran (2:1, 3 ml), added with(tert-butoxycarbonyl-methylene)triphenylphosphorane (176 mg, 0.467mmol), and stirred at 70° C. for 11 hours. The reaction mixture wasconcentrated, and the resulting residue was separated and purified bypreparative TLC (chloroform:methanol=100:5) to obtain Z-isomer of thetitle compound (68.7 mg, 51%) and E-isomer of the title compound (32.9mg, 29%) as pale yellow solids.

(Z-Isomer) Rf=Higher

¹H-NMR (CDCl₃) δ: 1.23 (2H, m), 1.32 (2H, m), 1.50 (9H, s), 3.43 (1H,m), 5.86 (1H, d, J=13.2Hz), 7.27 (1H, d, J=13.2Hz), 7.71 (1H, dd, J=11.5and 6.4Hz), 8.22 (1H, dd, J=10.0 and 9.1Hz), 9.36 (1H, s)

MS; m/z: 348 (MH⁺)

(E-Isomer) Rf=Lower

¹H-NMR (CDCl₃) δ: 1.11 (2H, m), 1.33 (2H, m), 1.52 (9H, s), 3.43 (1H,m), 7.14 (1H, d, J=15.8Hz), 7.39 (1H, d, J=15.8Hz), 7.70 (1H, dd, J=11.5and 6.3Hz), 7.88 (1H, s), 8.24 (1H, dd, J=10.5 and 8.5Hz)

MS; m/z: 348 (MH⁺)

(E) tert-Butyl(Z)-3-(1-cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-4-oxo-1,4-dihydro-3-quinolyl)-2-propenoate

(4-Isopropyl-1,3-thiazol-2-yl)methanol (31.1 mg, 0.198 mmol) wasdissolved in dimethylformamide (1 ml), added with 18-crown-6 (57.5 mg,0.218 mmol) and sodium hydride (95%, 5.5 mg, 0.218 mmol), and then themixture was stirred for 15 minutes under argon atmosphere. The reactionmixture was added with a solution of the tert-butyl(Z)-3-(1-cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydro-3-quinolyl)-2-propenoate(68.7 mg, 0.198 mmol) obtained in (D) in dimethylformamide (1 ml) andstirred at room temperature for 2 hours. Then, the reaction mixture wasadded with aqueous ammonium chloride and extracted with chloroform. Theorganic layer was dried over magnesium sulfate, and the solvent wasevaporated under reduced pressure. The obtained residue was purified bypreparative TLC (chloroform:methanol=100:1) to obtain the title compound(52.4 mg, 55%).

¹H-NMR (CDCl₃) δ: 1.15 (2H, m), 1.29 (2H, m), 1.33 (3H, d, J=6.8Hz),1.50 (9H, s), 3.11 (1H, m), 3.36 (1H, m), 5.58 (2H, s), 5.82 (1H, d,J=12.9Hz), 6.97 (1H, s), 7.27 (1H, d, J=12.9Hz), 7.55 (1H, d, J=6.8Hz),8.11 (1H, d, J=11.5Hz), 9.32 (1H, s)

(F)(Z)-3-(1-Cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-4-oxo-1,4-dihydro-3-quinolyl)-2-propeonicacid

The(Z)-3-(1-cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyl]-4-oxo-1,4-dihydro-3-quinolyl)-2-propenoate(52.4 mg, 0.108 mmol) obtained in (E) was dissolved in 4 N hydrochloricacid in dioxane (2 ml) and stirred overnight. The reaction mixture wasconcentrated, and the deposited solid was collected by filtration usingether to obtain the title compound (54.3 mg, quantitative) as paleyellow solid.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.36 (3H, d, J=7.10Hz), 1.53 (2H, m), 1.73 (2H,m), 3.18 (1H, m), 4.38 (1H, m), 5.90 (2H, s), 6.77 (1H, d, J=9.6Hz),7.26 (1H, s), 8.25 (1H, d, J=9.6Hz), 8.41 (2H, m), 9.79 (1H, s)

ES-MS; m/z: 427 (MH⁻)

Example 162(E)-3-(1-Cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-4-oxo-1,4-dihydro-3-quinolyl)-2-propeonicacid

(A) tert-Butyl(E)-3-(1-cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-4-oxo-1,4-dihydro-3-quinolyl)-2-propenoate

(4-Isopropyl-1,3-thiazol-2-yl)methanol (19.5 mg, 0.124 mmol) wasdissolved in dimethylformamide (1 ml), added with 18-crown-6 (35.8 mg,0.135 mmol) and sodium hydride (95%, 3.4 mg, 0.135 mmol), and stirredfor 15 minutes under argon atmosphere. The reaction mixture was addedwith a solution of tert-butyl(E)-3-(1-cyclopropyl-6,7-difluoro-4-oxo-1,4-dihydro-3-quinolyl)-2-propenoate(39.2 mg, 0.124 mmol) in dimethylformamide (1 ml) and stirred at roomtemperature for 3 hours. Then, the reaction mixture was added withaqueous ammonium chloride and extracted with chloroform. The organiclayer was dried over magnesium sulfate, and the solvent was evaporatedunder reduced pressure. The resulting residue was purified bypreparative TLC (chloroform:methanol=100:1) to obtain the title compound(44.7 mg, 82%).

¹H-NMR (CDCl₃) δ: 1.03 (2H, m), 1.30 (2H, m), 1.31 (3H, d, J=6.8Hz),1.51 (9H, s), 3.11 (1H, m), 3.36 (1H, m), 5.57 (2H, s), 6.96 (1H, s),7.12 (1H, d, J=15.8Hz), 7.37 (1H, d, J=15.8Hz), 7.53 (1H, d, J=6.8Hz),7.79 (1H, s), 8.10 (1H, d, J=11.2Hz)

(B)(E)-3-(1-Cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-4-oxo-1,4-dihydro-3-quinolyl)-2-propeonicacid

The(E)-3-(1-cyclopropyl-6-fluoro-7-[(4-isopropyl-1,3-thiazol-2-yl)methyloxy]-4-oxo-1,4-dihydro-3-quinolyl)-2-propenoate(44.7 mg, 0.0992 mmol) obtained in (A) was dissolved in 4 N hydrochloricacid in dioxane (2 ml) and stirred overnight. The reaction mixture wasconcentrated, and the deposited solid was collected by filtration usingether to obtain the title compound (32.9 mg, 83%) as pale yellow solid.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.14 (2H, m), 1.33 (3H, d, J=5.6Hz), 1.38 (2H,m), 3.15 (1H, m), 3.56 (1H, m), 5.66 (2H, s), 7.13 (1H, d, J=15.6Hz),7.17 (1H, s), 7.60 (1H, d, J=15.6Hz), 7.75 (1H, d, 6.9Hz), 8.04 (1H, d,J=11.2Hz), 8.25 (1H, s)

ES-MS; m/z: 429 (MH⁺)

Example 163(E)-3-[2-[3-(Aminocarbonyl)piperidino]-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A)tert-Butyl-(E)-3-[8-([4-(tert-butyl)-1,3-thiazol-2-yl]aminocarbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

DMF (28.1 μl, 0.36 mmol) was dissolved in methylene chloride (3 ml),added dropwise with oxalyl chloride (31.7 μl, 0.36 mmol) with icecooling and stirred for 15 minutes at the same temperature. The reactionsolution was added withN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxamide(50.0 mg, 0.15 mmol) and stirred at room temperature for 1 hour and 30minutes. The reaction mixture was added with a small amount of saturatedaqueous sodium hydrogencarbonate with ice cooling, extracted withchloroform at about pH 7 and dried over anhydrous sodium sulfate, andthe solvent was evaporated. The residue was dissolved in THF (10 ml),added with (tert-butoxycarbonylmethylene)triphenylphosphorane (82.0 mg,0.22 mmol) and stirred at room temperature for 13 hours. The solvent wasevaporated, and the residue was purified by preparative TLC (two plates,chloroform:methanol=20:1, v/v) to obtain the title compound (34.3 mg,50.2%).

¹H-NMR (DMSO-d₆) δ: 1.30 (9H, s), 1.45 (9H, s), 6.87 (1H, d, J=15.7Hz),6.82-6.91 (1H, m), 7.82 (1H, d, J=15.7Hz), 7.87-7.95 (2H, m), 9.07 (1H,d, J=7.6Hz).

MS; m/z: 471 (MH⁺).

(B) tert-Butyl(E)-3-[2-[3-(aminocarbonyl)piperidino]-8-([4-(tert-butyl)-1,3-thiazol-2-yl]aminocarbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

tert-Butyl(E)-3-[8-([4-(tert-butyl)-1,3-thiazol-2-yl]aminocarbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(67.3 mg, 0.14 mmol) was dissolved in DMF (20 ml), added with tosylchloride (40.9 mg, 0.21 mmol) and dimethylaminopyridine (26.2 mg, 0.21mmol), stirred at room temperature for 3 hours, added with nipecotamide(91.7 mg, 0.72 mmol), and stirred at room temperature for 15 hours. Thesolvent was evaporated, and the residue was purified by preparative PLC(chloroform:methanol=30:1, v/v) to obtain the title compound (53.9 mg,64.9% for the two steps) as an amorphous mixture of pale orange oilysubstance.

¹H-NMR (CDCl₃) δ: 1.31 (9H, s), 1.51 (9H, s), 1.50-1.85 (3H, m),1.96-2.03 (1H, m), 2.74-2.95 (2H, m), 3.19-3.28 (1H, m), 4.04 (1H, brd,J=12.7Hz), 4.41 (1H, brd, J=12.7Hz), 6.61 (1H, s), 6.76 (1H, brs),6.96(1H, brs), 7.07 (1H, d, J=15.4Hz), 7.44(1H, dd, J=7.6, 1.5Hz), 7.51(1H, d, J=15.4Hz), 7.97 (1H, brs), 8.97 (1H, d, J=7.6Hz).

(C)(E)-3-[2-[3-(Aminocarbonyl)piperidino]-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}-carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid (D 11-2168)

tert-Butyl(E)-3-[2-[3-(aminocarbonyl)piperidino]-8-([4-(tert-butyl)-1,3-thiazol-2-yl]aminocarbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(60.6 mg, 0.10 mmol) was dissolved in 4 N hydrochloric acid in dedioxane(6 ml) and stirred at room temperature for 3 hours. The solvent and theexcessive reagents were evaporated, and the residue was subjected toazeotropy with ether. The residue was purified by preparative PLC(chloroform:methanol=10:1, v/v) and lyophilized from dioxane to obtainthe title compound (35.8 mg, 65.4%) as orange powder.

m.p.: 206-223° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.24 (9H, s), 1.60-1.70 (2H, m), 1.72-1.81 (1H, m),1.94-2.01 (1H, m), 3.07-3.20 (2H, m), 3.40-3.50 (1H, m), 3.93 (1H, brd,J=12.8Hz), 4.14 (1H, brd, J=12.8Hz), 6.80-6.98 (2H, m), 6.93 (1H, d,J=15.5Hz), 7.31 (1H, brs), 7.44 (1H, d, J=15.5Hz), 7.62 (1H, d,J=6.6Hz), 8.22 (1H, brs), 8.90 (1H, d, J=7.3Hz).

IR (microscopic ATR): 3320, 3191, 2960, 2861, 1666, 1592, 1519, 1442cm⁻¹.

FAB/MS; m/z: 525 (MH⁺).

H-R FAB/MS: Calcd. for C₂₅H₂₈N₆O₅S: 525.1920. Found: 525.1880.

In a similar manner, the following compounds were synthesized.

Example 164(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3-[(dimethylamino)carbonyl]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

m.p.: 175-187° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.02 (3H, t, J=7.0Hz), 1.18 (3H, t, J=7.0Hz), 1.31(9H, s), 1.60-1.79 (3H, m), 1.82-1.89 (1H, m), 2.88-2.97 (1H, m),3.09-3.21 (2H, m), 3.38-3.55 (4H, m), 3.94 (1H, brd, J=12.9Hz), 4.10(1H, brd, J=13.2Hz), 6.86 (1H, brs), 6.94 (1H, d, J=15.4Hz), 7.43 (1H,d, J=15.5Hz), 7.65 (1H, d, J=6.1Hz), 8.12 (1H, brs), 8.91 (1H, d,J=7.6Hz).

IR (microscopic ATR): 2962, 2933, 2869, 1677, 1633, 1598, 1546, 1519,1442 cm⁻¹.

FAB/MS; m/z: 581 (MH⁺).

H-R FAB/MS: Calcd. for C₂₉H₃₆N₆O₅S: 581.2546. Found: 581.2526.

Example 165(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3-{[(methylamino)carbonyl]amino}piperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

m.p.: 220-225° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.40-1.52 (1H, m), 1.60-1.73 (1H, m),1.73-1.86 (1H, m), 1.86-1.96 (1H, m), 2.52 (3H, d, J=4.4Hz), 3.05-3.17(1H, m), 3.18-3.30 (1H, m), 3.53-3.46 (1H, m), 3.69-3.78 (1H, m),3.95-4.04 (1H, m), 3.66-3.72 (1H, m), 5.90-5.98 (1, m), 6.80-6.95 (1H,m), 6.92 (1H, d, J=15.5Hz), 7.44 (1H, d, J=15.5Hz), 7.60 (1H, d,J=5.9Hz), 8.19 (1H, brs), 8.89 (1H, d, J=7.6Hz).

IR (microscopic ATR): 3345, 2927, 2854, 1673, 1635, 1592, 1558, 1515,1436 cm⁻¹.

FAB/MS; m/z: 554 (MH⁺).

H-R FAB/MS: Calcd. for C₂₆H₃₁N₇O₅S: 554.2186. Found: 554.2151.

Example 166(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3-{[(dimethylamino)carbonyl]amino}piperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoic acid

m.p.: 194-200° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.53-1.72 (2H, m), 1.76-1.85 (1H, m),1.85-1.94 (1H, m), 2.77 (6H, s), 3.00-3.24 (2H, m), 3.57-3.69 (1H, m),3.81-3.90 (1H, m), 4.04-4.12 (1H, m), 6.09 (1H, d, J=7.1Hz), 6.80-6.98(1H, m), 6.92 (1H, d, J=15.7Hz), 7.43 (1H, d, J=15.7Hz), 7.60 (1H, d,J=7.3Hz), 8.22 (1H, brs), 8.89 (1H, d, J=7.4Hz).

IR (microscopic ATR): 2927, 2861, 1673, 1633, 1596, 1513, 1440 cm⁻¹.

FAB/MS; m/z: 568 (MH⁺).

H-R FAB/MS: Calcd. for C₂₇H₃₃N₇O₅S: 568.2342. Found: 568.2339.

Example 167(E)-3-[2-{3-[(Aminocarbonyl)amino]piperidino}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.39-1.52 (1H, m), 1.60-1.96 (3H, m),3.08-3.18 (1H, m), 3.40-3.65 (2H, m), 3.70-3.78 (1H, m), 3.97-4.03 (1H,m), 5.43 (2H, brs), 6.03 (1H, d, J=8.3Hz), 6.86 (1H, brs), 6.92 (1H, d,J=15.5Hz), 7.44 (1H, d, J=15.5Hz), 7.60 (1H, d, J=7.3Hz), 8.19 (1H,brs), 8.90 (1H, d, J=7.3Hz).

IR (microscopic ATR): 2958, 2925, 2856, 1668, 1594, 1540, 1519, 1436cm⁻¹.

FAB/MS; m/z: 540 (MH⁺).

H-R FAB/MS: Calcd. for C₂₅H₂₉N₇O₅S: 540.2029. Found: 540.2033.

Example 168(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3-{[(2-methoxyethyl)amino]carbonyl}piperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoic acid

m.p.: 183-190° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.58-1.82 (3H, m), 1.88-1.97 (1H, m),3.05-3.36 (7H, m), 3.23 (3H, s), 3.90-3.99 (1H, m), 4.08-4.15 (1H, m),6.87 (1H, brs), 6.93 (1H, d, J=15.4Hz), 7.42 (1H, d, J=15.4Hz), 7.60(1H, d, J=7.1Hz), 7.87-7.94 (1H, m), 8.22 (1H, brs), 8.90 (1H, d,J=7.3Hz).

IR (microscopic ATR): 3320, 2935, 2865, 1668, 1592, 1519, 1442 cm⁻¹.

FAB/MS; m/z: 583 (MH⁺).

H-R FAB/MS: Calcd. for C₂₈H₃₄N₆O₆S: 583.2339. Found: 583.2358.

Example 169(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[3-(hydroxymethyl)piperidino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

m.p.: 177-187° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.57-1.79 (1H, m), 1.79-1.83 (3H, m),2.88-2.98 (1H, m), 3.06-3.17 (1H, m), 3.30-3.43 (3H, m), 3.90-3.98 (1H,m), 4.10-4.19 (1H, m), 4.55 (1H, t, J=4.9Hz), 6.87 (1H, brs), 6.92 (1H,d, J=15.4Hz), 7.43 (1H, d, J=15.4Hz), 7.58 (1H, d, J=6.9Hz), 8.18 (1H,brs), 8.88 (1H, d, J=7.6Hz).

IR (microscopic ATR): 2925, 2856, 1673, 1590, 1521, 1440 cm⁻¹.

FAB/MS; m/z: 512 (MH⁺).

H-R FAB/MS: Calcd. for C₂₅H₂₉N₅O₅S: 512.1968. Found: 512.1969.

Example 170(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3-{[(2-hydroxyethyl)amino]carbonyl}piperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

m.p.: 173-190° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.59-1.84 (3H,m), 1.88-2.00 (1H, m),3.05-3.25 (3H, m), 3.88-3.98 (1H, m), 4.10-4.19 (1H, m), 4.60-4.72 (1H,m), 6.86 (1H, brs), 6.94 (1H, d, J=15.6Hz), 7.43 (1H, d, J=15.6Hz), 7.62(1H, d, J=7.1Hz), 7.86 (1H, t, J=4.9Hz), 8.23 (1H, brs), 8.91 (1H, d,J=7.6Hz).

IR (microscopic ATR): 3318, 2935, 2863, 1668, 1641, 1592, 1519, 1442cm⁻¹.

FAB/MS; m/z: 569 (MH⁺).

H-R FAB/MS: Calcd. for C₂₇H₃₂N₆O₆S: 569.2182. Found: 569.2160.

Example 171(E)-3-[2-(3-{[(Aminocarbonyl)oxy]methyl}piperidino)-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

m.p.: 167-178° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.58-1.72 (1H, m), 1.72-1.90 (2H, m),1.91-2.03 (1H, m), 2.92-3.01 (1H, m), 3.07-3.16 (1H, m), 3.30-3.42 (1H,m), 3.80-3.93 (3H, m), 4.08-4.14 (1H, m), 6.48 (2H, brs), 6.87 (1H,brs), 6.94 (1H, d, J=15.5Hz), 7.44 (1H, d, J=15.5Hz), 7.61 (1H, d,J=6.6Hz), 8.19 (1H, brs), 8.91 (1H, d, J=7.6Hz).

IR (microscopic ATR): 3673, 3345, 3181, 2967, 2900, 2865, 1673, 1592,1519, 1442, 1409 cm⁻¹.

FAB/MS; m/z: 555 (MH⁺).

H-R FAB/MS: Calcd. for C₂₆N₃₀N₆O₆S: 555.2026. Found: 555.2026.

Example 172N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-{3-[(aminocarbonyl)amino]piperidino}-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

m.p.: 260-310° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.40-1.52 (1H, m), 1.68-1.88 (2H, m),1.89-1.99 (1H, m), 2.93-3.30 (2H, m), 3.61-3.73 (1H, m), 3.84-3.93 (1H,m), 3.99-4.07 (1H, m), 5.51 (2H, brs), 6.11 (1H, d, J=8.3Hz), 6.88 (1H,brs), 7.44 (1H, d, J=15.9Hz), 7.63 (1H, d, J=6.9Hz), 7.83 (1H, d,J=15.9Hz), 8.22 (1H, brs), 8.94 (1H, d, J=7.3Hz).

IR (microscopic ATR): 3318, 3095, 2925, 2854, 1670, 1614, 1519, 1440cm⁻¹.

FAB/MS; m/z: 564 (MH⁺).

H-R FAB/MS: Calcd. for C₂₁H₂₉N₁₁O₃S: 564.2254. Found: 564.2285.

Example 173(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[3-({[2-(dimethylamino)ethyl]amino}carbonyl)piperidino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.31 (9H, s), 1.59-1.72 (2H, m), 1.72-1.86(1H, m), 1.86-1.98 (1H, m), 2.22 (6H, s), 2.32-2.45 (2H, m), 3.05-3.35(5H, m), 4.01 (1H, brd, J=12.4Hz), 4.09 (1H, brd, J=12.4Hz), 6.85 (1H,s), 6.92 (1H, d, J=15.4Hz), 7.42 (1H, d, J=15.4Hz), 7.61 (1H, d,J=7.6Hz), 7.80-7.90 (1H, m), 8.19 (1H, s), 8.89 (1H, d, J=7.3Hz).

IR (microscopic ATR): 2958, 2937, 2861, 1660, 1519, 1440 cm⁻¹.

m.p.: 177-187° C. (decomp.)

FAB/MS m/z: 596 (MH⁺).

HR-FAB/MS: 596.2646 (Calcd. for C₂₉H₃₇N₇O₅S: 596.2655).

Example 174(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3-{[3-(dimethylamino)propanoyl]amino}piperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.31 (9H, s), 1.43-1.60 (1H, m), 1.60-1.76(1H, m), 1.78-1.96 (2H, m), 2.27 (6H, s), 2.25-2.38 (1H, m), 2.59-2.71(1H, m), 3.05-3.25 (3H, m), 3.63-3.75 (2H, m), 3.75-3.87 (1H, m),3.87-3.98 (1H, m), 6.85 (1H, s), 6.92 (1H, d, J=15.4Hz), 7.44 (1H, d,J=15.4Hz), 7.61 (1H, dd, J=7.3, 2.0Hz), 8.13 (1H, brd, J=7.1Hz), 8.16(1H, d, J=2.0Hz), 8.90 (1H, d, J=7.3Hz).

IR (microscopic ATR): 2956, 2360, 1660, 1515, 1440 cm⁻¹.

m.p.: 177-197° C. (decomp.)

FAB/MS m/z: 596 (MH⁺).

HR-FAB/MS: 596.2646 (Calcd. for C₂₉H₃₇N₇O₅S: 596.2655).

Example 175(2-{[(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-carboxy-1-ethenyl]-4-oxo-4H-.pyrido[1,2-a]pyrimidin-2-yl}-3-piperidyl)carbonyl]aminoethyl)(trimethyl) ammoniumiodide

(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[3-({[2-(dimethylamino)ethyl]amino}carbonyl)piperidino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid (D11-4378, 15.4 mg, 0.026 mmol) was dissolved in DMF (1 ml), addeddropwise with methyl iodide (100 μl) and left in a refrigerator for 24hours. The solvent and the excessive reagents were evaporated, and theresidue was subjected to azeotropy with toluene and ether. The residuewas lyophilized from dioxane/water to obtain 20.6 mg (quantitative) ofthe title compound as yellow orange powder.

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.31 (9H, s), 1.60-1.75 (2H, m), 1.75-1.83(1H, m), 1.92-2.02 (1H, m), 3.09 (9H, s), 3.05-3.55 (7H, m), 3.90-3.98(1H, m), 4.07-4.14 (1H, m), 6.88 (1H, brs), 6.94 (1H, d, J=15.4Hz), 7.43(1H, d, J=15.4Hz), 7.64 (1H, d, J=7.1Hz), 8.18 (1H, s), 8.21 (1H, brt,J=5.4Hz), 8.92 (1H, d, J=7.3Hz).

IR(microscopic ATR) cm⁻¹: 3409, 2956, 2763, 2362, 1664, 1590, 1517,1444.

m.p.: 132-142° C. (decomp.)

FAB/MS m/z: 610 (M⁺).

HR-FAB/MS: 610.2803 (Calcd. for C₃₀H₄₀N₇O₅S: 610.2812).

In a similar manner, the following compounds were synthesized.

Example 176{3-[(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-carboxy-1-ethenyl-4-oxo-4H-pyrido-3-[1,2-a]pyrimidin-2-yl}-3-piperidyl)amino]-3-oxopropyl}(trimethyl)ammoniumiodide

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.31 (9H, s), 1.47-1.60 (1H, m), 1.60-1.77(1H, m), 1.80-2.00 (2H, m), 2.60-2.78 (2H, m), 3.04 (9H, s), 3.12-3.60(4H, m), 3.70-3.90 (2H, m), 3.92-4.02 (1H, m), 6.88 (1H, brs), 6.94 (1H,d, J=15.6Hz), 7.45 (1H, d, J=15.6Hz), 7.64 (1H, d, J=7.6Hz), 8.00-8.30(2H, m), 8.22 (1H, d, J=6.9Hz), 8.92 (1H, d, J=7.6Hz).

IR (microscopic ATR): 3019, 2981, 2807, 2773, 1670, 1521, 1457, 1403cm⁻¹.

m.p.: 117-124° C. (decomp.)

FAB/MS m/z: 610 (M⁺).

HR-FAB/MS: 610.2830 (Calcd. for C₃₀H₄₀N₇O₅S: 610.2812).

Example 177(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3-{[2-(dimethylamino)acetyl]amino}piperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoic acid

¹H-NMR (400 MHz, CDCl₃) δ: 1.39 (9H, s), 1.72-2.17 (4H, m), 2.08 (6H,s), 2.84 (1H, ABq, J=15.9Hz), 2.91 (1H, ABq, J=15.9Hz), 3.53-3.66 (1H,m), 3.69-4.00 (3H, m), 4.12-4.22 (1H, m), 6.66 (1H, s), 7.32 (1H, d,J=15.6Hz), 7.74 (1H, d, J=15.6Hz), 7.78 (1H, d, J=6.4Hz), 7.83 (1H, d,J=7.1Hz), 8.32 (1H, s), 9.09 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 3050, 2946, 2830, 2778, 1668, 1594, 1511, 1438.

m.p.: 193-203° C. (decomp.)

FAB/MS m/z: 582 (MH⁺).

HR-FAB/MS: 582.2492 (Calcd. for C₂₈H₃₅N₇O₅S: 582.2499).

Example 178(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-(3-{[2-(1,1,1-trimethylammonio)acetyl]amino}piperidino)-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

¹H-NMR (400 MHz, CD₃OD) δ: 1.33 (9H, s), 1.64-1.90 (2H, m), 1.90-2.08(2H, m), 3.32 (9H, s), 3.37-3.53 (2H, m), 3.60-3.76 (1H, m), 3.80-3.96(1H, m), 4.02-4.24 (3H, m), 6.75 (1H, s), 7.07 (1H, d, J=15.4Hz), 7.58(1H, d, J=15.4Hz), 7.63 (1H, d, J=7.6Hz), 8.02 (1H, s), 8.98 (1H, d,J=7.3Hz).

IR (ATR) cm⁻¹: 3193, 2958, 2854, 1662, 1554, 1515, 1440.

m.p.: 213-225° C. (decomp.)

FAB/MS m/z: 596 (MH⁺).

Anal. Calcd. for C₂₉H₃₇N₇O₅S.7H₂O: C, 48.25; H, 7.12; N, 13.58. Found:C, 47.85; H, 6.97; N, 13.36.

Example 179N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-((3S)-3-{[2-(dimethylamino)acetyl]amino}hexahydro-1-pyridinyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-((3S)-3-{[2-(dimethylamino)acetyl]amino}hexahydro-1-pyridinyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine -8-carboxyamideN⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(106.3 mg, 0.19 mmol) was suspended in acetonitrile (3 ml) and DMF (3ml), added dropwise with diphenyl chlorophosphate (79.0 μl, 0.38 mmol)and diisopropylethylamine (132.6 μl, 0.76 mmol) at −10° C. under argonatmosphere, stirred at the same temperature for 15 minutes, then addeddropwise with a solution ofN¹-[(3S)-hexahydro-3-pyridinyl]-2-(dimethylamino)acetamide (70.5 mg,0.38 mmol) in DMF (1 ml) and diisopropylethylamine (66.3 μl, 0.38 mmol)and heated to 80° C. for 1 hour with stirring. The reaction solution wasadded with a solution ofN¹-[(3S)-hexahydro-3-pyridinyl]-2-(dimethylamino)acetamide (70.5 mg,0.38 mmol) in DMF (1 ml) and diisopropylethylamine (66.3 μl, 0.38 mmol),further heated to 80° C. for 1 hour with stirring and then cooled. Thereaction mixture was added with saturated aqueous sodiumhydrogencarbonate and extracted with chloroform. The resulting organiclayer was washed with saturated brine and dried over anhydrous sodiumsulfate, and the solvent was evaporated. The residue was purified bypreparative TLC (chloroform:methanol=10:1) to obtain the title compound(111.5 mg, 80.7%) as yellow orange oily substance.

¹H-NMR (400 MHz, CDCl₃) δ: 1.37 (9H, s), 1.45-1.94 (4H, m), 2.20 (6H,s), 2.85-3.10 (1H, m), 3.12-3.24 (1H, m), 3.28-3.40 (1H, m), 3.42-3.68(2H, m), 3.78 (3H, s), 3.80-4.01 (1H, m), 4.07-4.20 (1H, m), 5.52 (2H,s), 6.56 (2H, s), 6.90 (2H, d, J=8.6Hz), 7.36 (2H, d, J=8.6Hz), 7.52(1H, d, J=7.1Hz), 7.72 (1H, d, J=15.5Hz), 7.90 (1H, d, J=15.5Hz), 8.01(1H, s), 8.88 (1H, d, J=7.3Hz).

(B)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-((3S)-3-{[2-(dimethylamino)acetyl]amino}hexahydro-1-pyridinyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-((3S)-3-{[2-(dimethylamino)acetyl]amino}exahydrol-pyridinyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(92.9 mg, 0.13 mmol) was dissolved in trifluoroacetic acid (20 ml) andstirred at room temperature for 62 hours, and the excessive reagent wasevaporated. The residue was subjected to azeotropy with toluene andether, and the residue was purified by preparative TLC(chloroform:methanol:water=8:3:0.5). The solvent was evaporated, and theresidue was powdered with ether and centrifuged. The supernatant wasremoved and the residue was dried under reduced pressure to obtain thetitle compound (28.9 mg, 30.1%) as orange powder.

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.72-1.85 (2H, m), 1.90-2.02(2H, m), 2.65 (6H, s), 3.46-3.75 (3H, m), 3.64 (2H, s), 3.75-3.82 (1H,m), 4.09-4.18 (1H, m), 6.74 (1H, s), 7.46 (1H, d, J=16.2Hz), 7.62 (1H,dd, J=7.6, 1.8Hz), 7.95 (1H, d, J=16.2Hz), 8.04 (1H, s), 9.00 (1H, d,J=7.6Hz).

IR (ATR) cm⁻¹: 3052, 2954, 2859, 1658, 1509, 1425.

m.p.: 201-207° C. (decomp.)

FAB/MS m/z: 606 (MH⁺).

HR-FAB/MS: 606.2714 (Calcd. for C₂₈H₃₅N₁₁O₃S: 606.2723).

Example 180N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-2-((3S)-3-{[2-(1,1,1-trimethylammonio)acetyl]amino}hexahydro-1-pyridinyl}-4H-pyrido[1,2-a]pyrimidine-8-carboxyamideformate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-((3S)-3-{[2-(dimethylamino)acetyl]amino}hexahydro-1-pyridinyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(D11-5061, 26.2 mg, 0.043 mmol) was dissolved in DMF (3 ml), addeddropwise with methyl iodide (300 μl) and left in a refrigerator for 62hours. The solvent and the excessive reagents were evaporated and theresidue was subjected to azeotropy with toluene and ether. The residuewas powdered by addition of ether, centrifuged and dried after thesupernatant was removed. The resulting powder was purified bypreparative TLC (chloroform:methanol=10:1, developed twice) and HPLC andlyophilized from dioxane and water to obtain the title compound (23.9mg, 30.1%) as orange powder.

HPLC Conditions

Column: CAPCELL PAK C18 SG120A, 5 μm, 30 mm φ×250 mm (SHISEIDO)

Mobile phase: H₂O:CH₃CN=70:30 (0.1% HCO₂H)

Detection wavelength: UV 254 nm

FR: 8 ml/min

Retention time: 12.0 min

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.70-1.87 (2H, m), 1.92-2.10(2H, m), 3.25 (9H, s), 3.46-3.58 (2H, m), 3.60-3.77 (2H, m), 4.08-4.23(3H, m), 6.75 (1H, s), 7.49 (1H, d, J=15.6Hz), 7.64 (1H, d, J=7.6Hz),7.96 (1H, d, J=15.6Hz), 8.06 (1H, s), 9.03 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 2954, 2923, 2854, 1733, 1670, 1548, 1519, 1444.

m.p.: 211-215° C. (decomp.)

FAB/MS. m/z: 620 (MH⁺).

HR-FAB/MS: 620.2824 (Calcd. for C₂₉H₃₇N₁₁O₃S: 620.2880).

Example 181(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2yl]amino}carbonyl)-4-oxo-2-[(3R)-3-({[(2-tetrahydro-1H-1-pyrrolylethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-[(3R)-3-({[(2-tetrahydro-1H-1-pyrrolylethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(2-chloroethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(0.51 g, purity: about 58%) was dissolved in DMF (5 ml) and addeddropwise with pyrrolidine (375 μl, 4.49 mmol). The reaction solution wasstirred under an argon flow at about 80° C. for 4 hours, further addedwith pyrrolidine (375 μl, 4.49 mmol) and stirred at about 80° C. for 3hours. The reaction mixture was further added with pyrrolidine (375μl,4.49 mmol), stirred at about 80° C. for 8 hours, added with saturatedaqueous sodium hydrogencarbonate and extracted with chloroform, theresulting organic layer was washed with saturated brine and dried overanhydrous sodium sulfate, and the solvent was evaporated. The residuewas purified by preparative TLC (chloroform:methanol=20:1) to obtain thetitle compound (235.8 mg, 75.7%) as a mixture of orange oily substanceand solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.33 (9H, a), 1.52 (9H, s), 1.58-1.75 (5H,m), 1.80-2.00 (3H, m), 2.25-2.78 (6H, m), 3.25-3.82 (6H, m), 4.80-4.95(1H, m), 5.75-5.86 (1H, m), 6.58 (1H, s), 7.07 (1H, d, J=15.4Hz), 7.48(1H, d, J=7.3Hz), 7.49 (1H, d, J=15.4Hz), 7.87 (1H, s), 8.96 (1H, d,J=7.3Hz).

(B)(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-[(3R)-3-({[(2-tetrahydro-1H-1-pyrrolylethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-[(3R)-3-({[(2-tetrahydro-1H-1-pyrrolylethyl)amino]carbonyl)oxy)hexahydro-1-pyridinyl]-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(235.8 mg, 0.34 mmol) was dissolved in 4 N hydrochloric acid solution indioxane (20 ml) and stirred at room temperature for 1 hour. Theexcessive reagent was evaporated, and the residue was subjected toazeotropy with toluene and ether. The residue was purified bypreparative TLC (chloroform:methanol=10:1) to obtain the title compound(197.3 mg, 91.0%) as yellow orange solid.

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.69-1.87 (2H, m), 1.90-2.18(6H, m), 3.05-3.45 (9H, m), 3.52-3.63 (1H, m), 3.90-4.00 (1H, m),4.08-4.19 (1H, m), 4.92-5.01 (1H, m), 6.74 (1H, s), 7.15 (1H, d,J=15.5Hz), 7.60 (1H, d, J=7.3Hz), 7.76 (1H, d, J=15.5Hz), 8.03 (1H, 8),8.97 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 2956, 2859, 1706, 1666, 1594, 1513, 1438.

m.p.: 162-182° C. (decomp.)

FAB/MS m/z: 638 (MH⁺).

HR-FAB/MS: 638.2772 (Calcd. for C₃₁H₃₉N₇O₆S: 638.2761).

Example 182(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[({[2-(1-methyltetrahydro-1H-1-pyrrolyl)ethyl]amino)carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid iodide

(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-[(3R)-3-({[(2-tetrahydro-1H-1-pyrrolylethyl)amino]carbonyl}oxy)hexahydro1-pyridinyl]-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid (D11-5127,99.2 mg, 0.16 mmol) was dissolved in DMF (10 ml), added dropwise withmethyl iodide (1 ml) and left in a refrigerator for 12 hours. Thesolvent and the excessive reagents were evaporated, and the residue wassubjected to azeotropy with toluene and ether. The residue was powderedby addition of ether and centrifuged. The supernatant was removed andthe residue was dried to obtain 124.9 mg of pale orange powder.

This compound (70 mg) was added with 4 N hydrochloric acid solution indioxane (4 ml) and stirred, and the excessive reagent was evaporated.This operation was further repeated twice, and the residue was subjectedto azeotropy with toluene and ether. The residue was purified bypreparative TLC (chloroform:methanol:water=8:3:0.5). The residue waspowdered by addition of ether and centrifuged. The supernatant wasremoved, and the residue was dried to obtain the title compound (52.3mg, 76.9%) as yellow orange powder.

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.60-1.73 (1H, m), 1.85-2.08(3H, m), 2.10-2.28 (4H, m), 3.13 (3H, s), 3.30-3.68 (11H, m), 3.83-4.07(2H, m), 6.73 (1H, s), 7.03 (1H, d, J=15.6Hz), 7.55 (1H, d, J=15.6Hz),7.57 (1H, dd, J=7.3, 2.0Hz), 7.96 (1H, s), 8.92 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 3384, 2958, 2861, 1700, 1662, 1596, 1508, 1438.

m.p.: 182-202° C. (decomp.)

FAB/MS m/z: 652 (MH⁺).

HR-FAB/MS: 652.2962 (Calcd. for C₃₂H₄₁N₇O₆S: 652.2917).

Anal. Calcd. for C₃₂H₄₁N₇O₆S.HI.4.8H₂O: C, 44.37; H, 6.00; N, 11.32.Found: C, 43.95; H, 5.51; N, 11.07.

Example 183N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3S)-3-({2-[1-(2-amino-2-oxoethyl)-1,1-dimethylammonio]acetyl}amino)hexahydro-1-pyridinyl]-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxyamidetrifluoroacetate

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3S)-3-({2-[-(2-amino-2-oxoethyl)-1,1-dimethylammonio]acetyl}amino)hexahydro-1-pyridinyl]3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamideiodide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-((3S)-3-{[2-(dimethylamino)acetyl]-amino}hexahydro-1-pyridinyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(468.3 mg, 0.65 mmol) was dissolved in DMF (30 ml), added withiodoacetamide (155.1 mg, 0.84 mmol) and stirred at room temperature for16 hours. The solvent was evaporated, and the residue was subjected toazeotropy with toluene and ether. The residue was purified bypreparative TLC (chloroform:methanol:water=8:3:0.1) to obtain the titlecompound (406.0 mg, 69.1%) as a mixture of orange oily substance andfoamy substance.

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.65-1.80 (2H, m), 1.84-2.04(2H, m), 3.42 (3H, s), 3.44 (3H, s), 3.40-3.58 (3H, m), 3.77 (3H, s),3.68-3.88 (3H, m), 4.03-4.12 (1H, m), 3.35-3.48 (3H, m), 4.50-4.62 (1H,m), 5.61 (1H, ABq, J=15.4Hz), 5.66 (1H, ABq, J=15.4Hz), 6.76 (1H, s),6.93 (2H, d, J=8.8Hz), 7.35 (2H, d, J=8.8Hz), 7.62-7.70 (1H, m), 7.69(1H, d, J=15.6Hz), 7.88 (1H, d, J=15.6Hz), 7.90 (1H, s), 8.06 (1H, s),9.01 (1H, d, J=7.3Hz).

(B)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3S)-3-({2-[1-(2-amino-2-oxoethyl)-1,1-dimethylammonio]acetyl}amino)hexahydro-1-pyridinyl]-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxyamidetrifluoroacetate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3S)-3-({2-[1-(2-amino-2-oxoethyl)-1,1-dimethylammonio]acetyl}amino)hexahydro-1-pyridinyl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamideiodide (401.3 mg, 4.41 mmol) was dissolved in TFA (80 ml) and stirred atroom temperature for 89 hours. The solvent was evaporated, and theresidue was subjected to azeotropy with toluene and ether. The residuewas purified by preparative TLC (chloroform:methanol water=8:3:0.5). Thesolvent was evaporated, and the residue was powdered by addition ofether and centrifuged. The supernatant was removed, and the residue wasdried under reduced pressure to obtain the title compound (172.6 mg,50.4%) as orange powder.

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.65-1.84 (2H, m), 1.88-2.04(2H, m), 3.35 (3H, s), 3.39 (3H, s), 3.30-3.53 (2H, m), 3.68-3.85 (2H,m), 4.05-4.16 (1H, m), 4.30 (1H, ABq, J=15.4Hz), 4.37 (1H, ABq,J=15.4Hz), 4.43 (2H, s), 6.75 (1H, s), 7.44 (1H, d, J=16.1Hz), 7.61 (1H,dd, J=7.5, 1.7Hz), 7.91 (1H, d, J=16.1Hz), 8.03 (1H, d, J=1.7Hz), 8.99(1H, d, J=7.8Hz).

IR (ATR) cm⁻¹: 3181, 3050, 2960, 2865, 1664, 1544, 1513, 1423.

m.p.: 180-203° C. (decomp.)

FAB/MS m/z: 663 (MH⁺).

HR-FAB/MS: 663.2910 (Calcd. for C₃₀H₃₉N₁₂O₄S: 663.2938).

Anal. Calcd. for C₃₀H₃₉N₁₂O₄S.C₂F₃O₂.2.6H₂O: C, 46.66; H, 5.41; N,20.41. Found: C, 47.23; H, 5.44; N, 19.77.

Example 184(E)-3-[2-((3R)-3-{[({2-[1-(2-Amino-2-oxoethyl)tetrahydro-1H-1-pyrrolyl]ethyl}amino)carbonyl]oxy}hexahydro-1-pyridinyl)-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.63-1.76 (1H, m), 1.83-1.96(1H, m), 1.96-2.27 (6H, m), 3.20-3.40 (2H, m), 3.44-3.99 (10H, m),4.18-4.27 (2H, m), 4.53-4.61 (1H, m), 6.75 (1H, s), 7.12 (1H, d,J=15.6Hz), 7.55-7.62 (1H, m), 7.59 (1H, d, J=15.6Hz), 8.02 (1H, s), 8.97(1H, d, J=7.6Hz).

IR (ATR) cm⁻¹: 2954, 2859, 1660, 1637, 1598, 1511, 1432.

m.p.: 188-199° C. (decomp.)

FAB/MS m/z: 695 (MH⁺).

HR-FAB/MS: 695.2957 (Calcd. for C₃₃H₄₂N⁸O₇S: 695.2975).

Example 185(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-((3S)-3-{[2-(1,1,1-trimethylammonio)acetyl]amino}hexahydro-1-pyridinyl)-4H-pyrido-[1,2-a]pyrimidin-3-yl]-2-propenoatehydrochloride

¹H-NMR (400 MHz, CD₃OD) δ: 1.34 (9H, s), 1.67-1.85 (2H, m), 1.85-2.05(2H, m), 3.33 (9H, s), 3.40-3.52 (2H, m), 3.52-3.68.(1H, m), 3.75-3.86(1H, m), 4.05-4.15.(1H, m), 4.14 (1H, ABq, J=15.1Hz), 4.23 (1H, d,J=15.1Hz), 6.72 (1H, s), 7.04 (1H, d, J=15.6Hz), 7.45 (1H, d, J=15.6Hz),7.56 (1H, d, J=7.6Hz), 7.94 (1H, s), 8.90 (1H, d, J=7.6Hz).

IR (ATR) cm⁻¹: 3345, 3189, 3046, 2956, 2861, 1664, 1596, 1540, 1513,1438.

m.p.: 207-217° C. (decomp.)

FAB/MS m/z: 596 (MH⁺).

HR-FAB/MS: 596.2672 (Calcd. for C₂₉H₃₇N₇O₅S: 596.2655).

Anal. Calcd. for C₂₉H₃₇N₇O₅S.HCl.3.9H₂O: C, 49.59; H, 6.57; N, 13.96.Found: C, 49.56; H, 6.05; N, 13.45.

Example 186(E)-3-[2-[(3S)-3-({2-[1-(2-Amino-2-oxoethyl)-1,1-dimethylammonio]acetyl}amino)hexahydro-1-pyridinyl]-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoatehydrochloride

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.68-1.83 (2H, m), 1.88-2.01(2H, m), 3.43 (3Hi s), 3.46 (3H, s), 3.40-3.80 (4H, m), 4.05-4.14 (1H,m), 4.36-4.52 (4H, m), 6.76 (1H, s), 7.08 (1H, d, J=15.6Hz), 7.44 (1Hd,J=15.6Hz), 7.61 (1H, dd, J=7.4, 1.5Hz), 8.03 (1H, d, J=1.5Hz), 8.97 (1H,d, J=7.4Hz).

IR (ATR) cm⁻¹: 3189, 2958, 2859, 1660, 1542, 1515, 1438.

m.p.: 185-197° C. (decomp.)

FAB/MS m/z: 639 (MH⁺).

HR-FAB/MS: 639.2697 (Calcd. for C₃₀H₃₈N₈O₆S: 639.2713).

Anal. Calcd. for C₃₀H₃₈N₈O₆S.2HCl.5.4H₂O: C, 44.54; H, 6.33; N, 13.85;S, 3.96. Found: C, 45.09; H, 6.00; N, 13.00; S, 3.90.

Example 187(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-((3R)-3-{[2-(1,1,1-trimethylammonio)acetyl]amino}hexahydro-1-pyridinyl)-4H-pyrido-[1,2-a]pyrimidin-3-yl]-2-propenoatehydrochloride

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.68-1.85 (2H, m), 1.90-2.02(2H, m), 3.32 (9H, s), 3.45-3.57 (2H, m), 3.57-3.68 (1H, m), 3.72-3.80(1H, m), 4.08-4.17 (1H, m), 4.11 (1H, ABq, J=15.1Hz), 4.23 (1H, ABq,J=15.1Hz), 6.75 (1H, s), 7.10 (1H, d, J=15.7Hz), 7.49 (1Hd, J=15.7Hz),7.62 (1H, d, J=7.3Hz), 8.02 (1H, s), 8.97 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 3199, 3050, 2954, 2929, 2857, 1664, 1596, 1540, 1513,1438.

m.p.: 210-220° C. (decomp.)

FAB/MS m/z: 596 (MH⁺).

HR-FAB/MS: 596.2660 (Calcd. for C₂₉H₃₇N₇O₅S: 596.2655).

Example 1882-(1-{2-[((3S)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)amino]-2-oxoethyl}-1,1-dimethylammonio)acetatetrifluoroacetate

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.68-1.82 (2H, m), 1.90-2.03(2H, m), 3.36 (6H, s), 3.25-2.50 (2H, m), 3.70-3.80 (1H, m), 3.83-3.92(1H, m), 3.97-4.09 (2H, m), 4.09-4.18 (1H, m), 4.39 (1H, ABq, J=14.6Hz),4.54 (1H, ABq, J=14.6Hz), 6.74 (1H, s), 7.53 (1H, d, J=16.1Hz), 7.63(1H, d, J=7.3Hz), 7.92 (1H, d, J=16.1Hz), 8.10 (1H, s), 9.01 (1H, d,J=7.3Hz).

IR (ATR) cm⁻¹: 3226, 3062, 2967, 2869, 1668, 1635, 1544, 1509, 1438,1403.

m.p.: 184-238° C. (decomp.)

FAB/MS m/z: 664 (MH⁺).

HR-FAB/MS: 664.2796 (Calcd. for C₃₀H₃₇N₁₁O₅S: 664.2778).

Anal. Calcd. for C₃₀H₃₇N₁₁O₅S.3.6C₂HF₃O₂.5.8H₂O: C, 37.91; H, 4.46; N,13.07; F, 17.41. Found: C, 37.46; H, 3.63; N, 12.39; F, 16.93.

Example 189(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(2-hydroxyethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(2-hydroxyethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl (E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate (204.7 mg,0.37 mmol) was dissolved in methylene chloride (20 ml), added withN,N′-carbonyldiimidazole (240.0 mg, 1.48 mmol) and stirred at roomtemperature for 1 hour and 30 minutes. The reaction mixture was washedwith water and saturated brine and dried over anhydrous sodium sulfate,and the solvent was evaporated to obtain 443.3 mg of a crude product.

The crude product was dissolved in THF (15 ml), and the atmosphere inthe system was replaced with nitrogen. The solution was added dropwisewith triethylamine (154.7 μl, 1.11 mmol) and 2-aminoethanol (44.6 μl,0.74 mmol) and stirred at room temperature for 14 hours. The solvent andthe excessive reagents were evaporated. Then the residue was subjectedto azeotropy with ether, added with chloroform, washed successively with1 N aqueous hydrochloric acid, saturated aqueous sodiumhydrogencarbonate and saturated brine and dried over anhydrous magnesiumsulfate, and the solvent was evaporated. The residue was purified bypreparative TLC (hexane:ethyl acetate=1:2) to obtain the title compound(182.4 mg, 77.0%) as a mixture of orange yellow oily substance and foamysubstance.

¹H-NMR (400 MHz, CDCl₃) δ: 1.33 (9H, s), 1.51 (9H, s), 1.60-1.68 (1H,m), 1.78-2.07 (4H, m), 3.20-3.61 (5H, m), 3.61-3.95 (3H, m), 4.03-4.17(1H, m), 4.90-4.99 (1H, m), 5.67-5.77 (1H, m), 6.59 (1H, s), 7.08 (1H,d, J=15.6Hz), 7.47 (1H, d, J=7.3Hz), 7.54 (1H, d, J=15.6Hz), 7.91 (1H,s), 8.96 (1H, d, J=7.3Hz).

(B)(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(2-hydroxyethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid

tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(2-hydroxyethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl}-2-propenoate(182.4 mg, 0.28 mmol) was dissolved in 4 N hydrochloric acid solution indioxane (20 ml) and stirred at room temperature for 3 hours. The solventand the excessive reagent were evaporated under reduced pressure, andthe residue was subjected to azeotropy with toluene and ether. Theresidue was purified by preparative TLC (chloroform:methanol=10:1) toobtain 114.2 mg of a compound. This compound (112.0 mg) was powdered byaddition of ether and centrifuged. The supernatant was removed, and theresidue was dried under reduced pressure to obtain the title compound(97.2 mg, 58.4%) as yellow orange powder.

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.63-1.76 (1H, m), 1.80-1.92(1H, m), 1.92-2.10 (2H, m), 3.15-3.24 (2H, m), 3.30-3.38 (1H, m),3.46-3.60 (3H, m), 3.72-3.90 (3H, m), 6.74 (1H, s), 7.02 (1H, d,J=15.6Hz), 7.57 (1H, dd, J=7.3, 1.7Hz), 7.63 (1H, d, J=15.6Hz), 8.01(1H, d, J=1.7Hz), 8.93 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 3444, 3369, 2954, 2871, 1710, 1675, 1585, 1515, 1432.

m.p.: 210-220° C. (decomp.)

FAB/MS m/z: 585 (MH⁺).

HR-FAB/MS: 585.2118 (Calcd. for C₂₇H₃₂N₆O₇S: 585.2131).

Anal. Calcd. for C₂₇H₃₂N₆O₇S.H₂O: C, 53.81; H, 5.69; N, 13.94. Found: C,54.13; H, 5.57; N, 13.84.

Example 190(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3S)-3-(glycoloylamino)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A) tert-Butyl(E)-3-[2-((3S)-3-{[2-(acetyloxy)acetyl]amino}hexahydro-1-pyridinyl)-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

tert-Butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(305.6 mg, 0.65 mmol) was substantially dissolved in DMF (50 ml), addedwith tosyl chloride (185.7 mg, 0.97 mmol) and dimethylaminopyridine(158.7 mg, 1.29 mmol) and stirred at room temperature for 4 hours. Thereaction solution was added with triethylamine (271.8 μl, 1.95 mmol) anda solution of 2-[(3S)-hexahydro-3-pyridinylamino]-2-oxoethylacetate (390mg, 1.95 mmol) in DMF (1 ml) and stirred at room temperature for 15hours. The solvent was evaporated, and the residue was subjected toazeotropy with toluene. The residue was purified by preparative TLC(chloroform:methanol=30:1, developed twice) and preparative TLC(hexane:ethyl acetate=1:2) to obtain the title compound (62.7 mg, 14.8%)as a mixture of yellow orange oily substance and foamy substance.

¹H-NMR (400 MHz, CDCl₃) δ: 1.34 (9H, s), 1.52 (9H, s), 1.60-2.00 (4H,m), 2.16 (3H, s), 3.50-3.73 (3H, m), 3.85-3.97 (1H, m), 4.20-4.30 (1H,m), 4.51 (1H, ABq, J=14.5Hz), 4.72 (1H, ABq, J=14.5Hz), 6.56 (1H, s),7.10 (1H, d, J=15.6Hz), 7.44 (1H, d, J=15.6Hz), 7.58 (1H, d, J=7.3Hz),8.07 (1H, s), 9.01 (1H, d, J=7.3Hz).

(B) tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3S)-3-(glycoloylamino)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl(E)-3-[2-((3S)-3-([2-(acetyloxy)acetyl]amino}hexahydro-1-pyridinyl)-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(62.7 mg, 0.096 mmol) was dissolved in THF (6 ml), added dropwise with 1N aqueous sodium hydroxide (192 μl, 0.19 mmol) and stirred for 17 hours.The solvent was evaporated, and then the residue was added withsaturated aqueous sodium hydrogencarbonate, and extracted withchloroform. The organic layer was washed with saturated brine and driedover anhydrous magnesium sulfate. The solvent was evaporated, and theresidue was purified by preparative TLC (chloroform:methanol=20:1) toobtain the title compound (49.7 mg, 84.7%) as a mixture of orange yellowoily substance and an amorphous substance.

¹H-NMR (400 MHz, CDCl₃) δ: 1.32 (9H, s), 1.51 (9H, s), 1.55-1.82 (5H,m), 1.97-2.07 (1H, m), 3.25-3.40 (2H, m), 3.77-3.95 (2H, m), 4.15 (1H,ABq, J=16.1Hz), 4.28 (1H, ABq, J=16.1Hz), 4.28-4.38 (1H, m), 6.60 (1H,s), 7.14 (1H, d, J=15.6Hz), 7.48 (1H, d, J=15.6Hz), 7.60 (1H, dd, J=7.3,1.7Hz), 8.07 (1H, d, J=7.6Hz), 8.13 (1H, d, J=1.7Hz), 9.02 (1H, d,J=7.3Hz).

(C)(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3S)-3-(glycoloylamino)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

tert-Butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino)carbonyl)-2-[(3S)-3-(glycoloylamino)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate(49.7 mg, 0.081 mmol) was dissolved in 4 N hydrochloric acid solution indioxane (5 ml) and stirred at room temperature for 19 hours. The solventand excessive reagents were evaporated, and the residue was subjected toazeotropy with toluene and ether. The residue was purified bypreparative TLC (chloroform:methanol=10:1). The solvent was evaporated,and the residue was powdered by addition of ether and centrifuged. Thesupernatant was removed, and the residue was dried under reducedpressure to obtain the title compound (45.0 mg, 99.7%) as yellow orangepowder.

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.65-2.02 (4H, m), 3.45-3.56(1H, m), 3.61-3.82 (3H, m), 3.90-3.97 (1H, m), 3.97 (2H, d, J=3.2Hz),4.02-4.10 (1H, m), 6.75 (1H, s), 7.03 (1H, d, J15.6Hz), 7.58 (1H, d,J=15.6Hz), 7.59 (1H, dd, J=7.3, 1.4Hz), 8.06 (1H, d, J=1.4Hz), 8.96 (1H,d, J=7.3Hz).

IR (ATR) cm⁻¹: 2952, 2859, 1664, 1511, 1438.

m.p.: 187-195° C. (decomp.)

FAB/MS m/z: 555 (MH⁺).

HR-FAB/MS: 555.2032 (Calcd. for C₂₆H₃₀N₆O₆S: 555.2026).

Anal. Calcd. for C₂₆H₃₀N₆O₆S.3.5H₂O: C, 50.56; H, 6.04; N, 13.61. Found:C, 50.66; H, 5.22; N, 12.78.

Example 1912-{1-[2-(4-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperazino)-2-oxoethyl]-1,1-dimethylammonio}acetate

(A)[2-(tert-Butoxy)-2-oxoethyl]{2-[4-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}-carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperazino]-2-oxoethyl}dimethylammoniumbromide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-4-[2-(dimethylamino)acetyl]piperazino}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl}-4-oxo-4H-pyrido-[1,2-a]pyrimidine-8-carboxamide(192.7 mg, 0.27 mmol) was dissolved in DMF (10 ml), added dropwise withtert-butyl bromoacetate (80.0 μl, 0.54 mmol) and stirred at roomtemperature for 63 hours. The solvent was evaporated, and the residuewas subjected to azeotropy with toluene and ether and purified bypreparative TLC (chloroform:methanol:water=8:3:0.5) to obtain the titlecompound (213.2 mg, 86.8%) as a mixture of yellow orange oily substanceand an amorphous substance.

¹H-NMR (400 MHz, CD₃OD) δ: 1.33 (9H, s), 1.52 (9H, s), 3.50 (6H, s),3.50-3.78 (10H, m), 3.74 (3H, s), 4.65 (2H, s), 5.57 (2H, s), 6.66 (1H,s), 6.89 (2H, d, J=8.7Hz), 7.30 (2H, d, J=8.7Hz), 7.55 (1H, d, J=7.3Hz),7.56 (1H, d, J=15.5Hz), 7.75 (1H, d, J=15.5Hz), 7.89 (1H, s), 8.86 (1H,d, J=7.3Hz).

(B)2-{1-[2-(4-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperazino)-2-oxoethyl]-1,1-dimethylammonio}acetate

[2-(tert-Butoxy)-2-oxoethyl]{2-[4-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperazino]-2-oxoethyl}dimethylammoniumbromide (213.2 mg, 0.24 mmol) was dissolved in 4 N hydrochloric acidsolution in dioxane (21 ml) and stirred at room temperature for 17hours. The solvent and excessive reagents were evaporated, and theresidue was subjected to azeotropy with toluene and ether. The residuewas purified by preparative TLC (chloroform:methanol:water=8:3:0.5) toobtain 170.4 mg of carboxylic acid compound as a mixture of yelloworange oily substance and an amorphous substance.

This compound was dissolved in TFA (50 ml), heated at 60° C. withstirring for 3 hours and cooled. Then TFA was evaporated, and theresidue was subjected to azeotropy with toluene and ether. The residuewas purified by preparative TLC (chloroform:methanol:water=8:3:0.5). Thepurified product was suspended in methanol and water, neutralized byaddition of 1 N aqueous sodium hydroxide and loaded on HP-20 (washedwith water and then eluted with methanol:water=7:3) for desalting. Theproduct was further purified by HPLC and preparative TLC(chloroform:methanol:water=8:3:0.5), powdered by addition of ether andcentrifuged. The supernatant was removed, and the residue was driedunder reduced pressure to obtain the title compound (47.1 mg, 30.8% forthe two steps) as orange powder.

HPLC conditions

Column: CAPCELL PAK C18 SG 120A, 5 μm, 30 mm φ×250 mm (SHISEIDO)

Mobile phase: MeOH:H₂O=50:50

Detection wavelength: UV 254 nm

FR: 12 ml/min

Retention time: 14.0 min

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 3.44 (6H, s), 3.38-3.47 (2H,m), 3.57-3.70 (4H, m), 3.65 (2H, s), 3.72-3.80 (2H, m), 4.20 (2H, s),6.70 (1H, s), 7.44 (1H, d, J=16.1Hz), 7.59 (1H, dd, J=7.4, 1.7Hz), 7.91(1H, d, J=16.1Hz), 7.95 (1H, s), 8.93 (1H, d, J=7.4Hz).

IR (ATR) cm⁻¹: 2964, 2867, 1916, 1652, 1621, 1548, 1509, 1465, 1448,1434.

m.p.: 230-245° C. (decomp.)

FAB/MS m/z: 650 (MH⁺).

HR-FAB/MS: 650.2629 (Calcd. for C₂₉H₃₅N₁₁O₅S: 650.2622).

Anal. Calcd. for C₂₉H₃₅N₁₁O₅S.3.6H₂O: C, 48.74; H, 5.95; N, 21.56.Found: C, 49.31; H, 5.49; N, 21.01.

Example 1922-{1-[2-(4-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-1,4-diazepan-1-yl)-2-oxoethyl]-1,1-dimethylammonio}acetate

[2-(tert-Butoxy)-2-oxoethyl]{2-[4-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}-carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-1,4-diazepan-1-yl]-2-oxoethyl}dimethylammoniumbromide (528.9 mg, 0.57 mmol) was dissolved in 4 N hydrochloric acidsolution in dioxane (53 ml) and stirred at room temperature for 33hours. The solvent and excessive reagents were evaporated, and theresidue was subjected to azeotropy with toluene and ether. The residuewas purified by preparative TLC (chloroform:methanol:water=8:3:0.1) toobtain 481.6 mg of carboxylic acid compound as a mixture of orange oilysubstance and foamy substance.

This compound was dissolved in TFA (100 ml), heated to 60° C. for 2hours with stirring and cooled. Then TFA was evaporated, and the residuewas subjected to azeotropy with toluene and ether and purified bypreparative TLC (chloroform:methanol:water=8:3:0.5). The product wassubstantially dissolved in water, neutralized by adding 1 N aqueoussodium hydroxide, loaded on HP-20 (washed with water and then elutedwith methanol:water=7:3) for desalting. The residue was purified byHPLC, powdered by addition of ether and centrifuged. The supernatant wasremoved, and the residue was dried under reduced pressure to obtain thetitle compound (96.0 mg, 30.8% for the two steps) as orange powder.

HPLC Conditions

Column: Develosil Combi-PR-5, 5 μm, 20×100 mm (NOMURA CHEMICAL CO.,LTD.)

Mobile phase: H₂O (0.1% HCO₂H):CH₃CN (0.1% HCO₂H)=69:31→40:60 (4 min)

Detection wavelength: UV 254 nm

FR: 25 ml/min

Retention time: 3.3 min

¹H-NMR (400 MHz, CD₃OD) δ: 1.35, 1.35 (total 9H, s), 1.92-2.02 (0.8H,m), 2.12-2.22 (1.2H, m), 3.82,3.84 (total 6H, s), 3.60-3.72 (2H, m),3.75-3.89 (4H, m), 3.97-4.10 (2H, m), 4.15, 4.22 (total 2H, s), 4.90,4.93 (total 2H, s), 6.73, 6.74 (total 1H, s), 7.49 (1H, d, J=16.1Hz),7.55, 7.59 (total 1H, dd, J=7.5, 1.7Hz), 7.81, 7.83 (total 1H, d,J=16.1Hz), 8.01, 8.07 (total 1H, d, J=1.7Hz), 8.11 (1H, s), 8.95, 8.97(total 1H, d, J=7.5Hz).

IR (ATR) cm⁻¹: 2958, 2867, 1652, 1515, 1425.

m.p.: 219-225° C. (decomp.)

FAB/MS m/z: 664 (MH⁺).

HR-FAB/MS: 664.2764 (Calcd. for C₃₀H₃₇N₁₁O₅S: 664.2778).

Anal. Calcd. for C₃₀H₃₇N₁₁O₅S.HCO₂H.0.3H₂O: C, 52.06; H, 5.58; N, 21.54.Found: C, 52.46; H, 5.93; N, 21.18.

Example 1932-{1-[2-(4-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-cis-2,6-dimethylhexahydro-1-pyrazinyl)-2-oxoethyl]-1,1-dimethylammonio}acetate

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-{4-[2-(dimethylamino)acetyl]-cis-3,5-dimethylhexahydro-1-pyrazinyl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(1.00 g, 1.79 mmol) was suspended in acetonitrile (20 ml) and DMF (20ml), added dropwise with diisopropyl ethylamine (1.25 ml, 7.16 mmol) anddiphenyl chlorophosphate (742.0 μl, 3.58 mmol) at −10° C. under argonatmosphere, stirred at the same temperature for 15 minutes, addeddropwise with cis-2,6-dimethylpiperazine (409.0 mg, 3.58 mmol) anddiisopropylethylamine (624.0 μl, 3.58 mmol), heated at 80° C. for 1 hourand 30 minutes with stirring and cooled. Then the reaction mixture wasdiluted with chloroform, washed with saturated aqueous sodiumhydrogencarbonate and saturated brine and dried over anhydrous sodiumsulfate, and the solvent was evaporated. The residue was purified bysilica gel column chromatography(chloroform→chloroform:methanol=30:1→10:1) to obtain 1.27 g of the2-substituted compound containing impurities as a mixture of orange oilysubstance and solid.

This compound (1.18 g) was suspended in THF (100 ml), added withN,N-dimethylglycine (0.93 g, 9.01 mmol), di-2-pyridylcarbonate (2.73 g,12.6 mmol) and dimethylaminopyridine (1.10 g, 9.01 mmol) under argonatmosphere, and heated to 60° C. for 7 hours with stirring. The solventwas evaporated, and the residue was added with saturated aqueous sodiumhydrogencarbonate, and extracted with ethyl acetate andchloroform/methanol (10:1); The organic layer was washed with saturatedbrine and dried over anhydrous sodium sulfate. The solvent wasevaporated, and the residue was purified by silica gel columnchromatography (chloroform→chloroform:methanol=30:1→20:1) andpreparative TLC (chloroform:methanol=10:1, developed twice) and(chloroform:ethyl acetate=2:1, developed seven times) to obtain thetitle compound (76.7 mg, 6.2% for the three 3 steps) as yellow orangepowder.

¹H-NMR (400 MHz, CDCl₃) δ: 1.15-1.45 (6H, m), 1.37 (9H, s), 2.28 (6H,s), 2.90-3.40 (6H, m), 3.50-3.63 (2H, m), 3.79 (3H, s), 5.54 (2H, s),6.58 (1H, s), 6.91 (2H, d, J=8.5Hz), 7.38 (2H, d, J=8.5Hz), 7.52 (1H, d,J=7.5Hz), 7.86 (1H, d, J=15.4Hz), 7.97 (1H, d, J=15.4Hz), 8.03 (1H, s),8.94 (1H, d, J=7.5Hz).

(B)[2-(tert-Butoxy)-2-oxoethyl]{2-[4-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}-carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)]-cis-2,6-dimethylhexahydro-1-pyrazinyl}-2-oxoethyl}dimethylammoniumbromide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-{4-[2-(dimethylamino)acetyl]-cis-3,5-dimethylhexahydro-1-pyrazinyl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(76.7 mg, 0.10 mmol) was dissolved in DMF (5 ml), added dropwise withtert-butyl bromoacetate (30.6,μl, 0.21 mmol) and stirred at roomtemperature for 20 hours. The solvent was evaporated, and the residuewas subjected to azeotropy with toluene and ether, and the residue waspurified by preparative TLC (chloroform:methanol:water=8:3:0.1) toobtain the title compound (51.9 mg, 53.5%) as a mixture of orange yellowoily substance and an amorphous substance.

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.37-1.45 (3H, m), 1.50-1.58(3H, m), 1.51 (9H, s), 3.43 (2H, s), 3.47 (6H, s), 3.76 (3H, s),3.96-4.15 (3H, m), 4.50-4.60 (1H, m), 4.60-5.00 (4H, m), 5.62 (2H, s),6.72 (1H, s), 6.92 (2H, d, J=8.8Hz), 7.34 (2H, d, J=8.8Hz), 7.65 (1H, d,J=7.4Hz), 7.77 (1H, d, J=15.4Hz), 7.89 (1H, d, J=15.4Hz), 8.03 (1H, s),8.99 (1H, d, J=7.6Hz).

(C)2-{1-[2-(4-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}cis-2,6-dimethylhexahydro-1-pyrazinyl)-2-oxoethyl]-1,1-dimethylammonio}acetate

[2-(tert-Butoxy)-2-oxoethyl]{2-[4-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)]-cis-2,6-dimethylhexahydro-1-pyrazinyl}-2-oxoethyl}-dimethylammoniumbromide (51.9 mg, 0.056 mmol) was dissolved in 4 N hydrochloric acidsolution in dioxane (5 ml), stirred at room temperature for 18 hours,added with 4 N hydrochloric acid solution in dioxane (5 ml) and stirredat room temperature for 24 hours. The solvent and excessive reagentswere evaporated, and the residue was subjected to azeotropy with tolueneand ether. The residue was purified by preparative TLC(chloroform:methanol:water=8:3:0.1) to obtain 29.4 mg of a carboxylicacid compound as yellow orange solid and recover 6.1 mg of the startingmaterial. The recovered starting material was treated in the same manneras above to obtain 4.7 mg of the carboxylic acid compound as yelloworange solid.

The carboxylic acid compound (34.1 mg) was dissolved in TFA (10 ml),heated to 60° C. with stirring for 2 hours and cooled. Then TFA wasevaporated, and the residue was subjected to azeotropy with toluene andether, and the residue was purified by preparative TLC(chloroform:methanol:water=8:3:0.5) and HPLC. The product was powderedby addition of ether and centrifuged. The supernatant was removed, andthe residue was dried under reduced pressure to obtain the titlecompound (9.7 mg, 25.8% for the two steps) as orange powder.

HPLC Conditions

Column: Develosil Combi-PR-5, 5 μm, 20×100 mm (NOMURA CHEMICAL CO.,LTD)

Mobile phase: H₂O (0.1% HCO₂H): CH₃CN (0.1% HCO₂H)=67:33→37:63 (4 min)

Detection wavelength: UV 254 nm

FR: 25 ml/min

Retention time: 3.35 min

¹H-NMR (400 MHz, CD₃OD) δ: 1.35 (9H, s), 1.35-1.62 (6H, m), 3.43 (6H,s), 3.35-3.52 (4H, m), 4.04-4.18 (3H, m), 4.24-4.39 (1H, m), 4.85-4.95(2H, m), 6.73 (1H, s), 7.64 (1H, dd, J=7.3, 2.0Hz), 7.68 (1H, d,J=16.0Hz), 7.89 (1H, d, J=16.0Hz), 8.07 (1H, d, J=2.0Hz), 8.10 (1H, s),9.00 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 3666, 2964, 2863, 2387, 2325, 2167, 2098, 1930, 1635,1546, 1511, 1502, 1432.

m.p.: 239-253° C. (decomp.)

FAB/MS m/z: 678 (MH⁺).

Anal. Calcd. for C₃₁H₃₉N₁₁O₅S.HCO₂H.2H₂O: C, 50.58; H, 5.97; N, 20.28.Found: C, 50.36; H, 5.96; N, 19.96.

Example 194 (E)3-[2-[4-((2S)-2-Amino-5-{[amino(imino)methyl]amino}pentanoyl)piperazino]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A)(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester

A solution of N,N-dimethylformamide (561 μl, 7.25 mmol) dissolved inmethylene chloride (60.0 ml) was added with oxalyl chloride (632 μl,7.25 mmol) with ice cooling and stirred at room temperature for 15minutes. This suspension was added withN⁸-[4-(t-butyl)-1,3-thiazol-2-yl]-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxyamide(1.00 g, 2.90 mmol) and stirred at room temperature for 1 hour. Thereaction was terminated with saturated aqueous sodium hydrogencarbonate,and the solution was adjusted to pH 5 with 1 N hydrochloric acid. Thissolution was extracted with chloroform (five times), and the combinedorganic layer was concentrated under reduced pressure. The resultingresidue was dissolved in a mixed solvent of tetrahydrofuran (500 ml) andN,N-dimethylformamide (20 ml), added with (t-butoxycarbonylmethylene)triphenylphosphorane (1.64 g, 4.34 mmol) and stirred overnight. Thereaction mixture was concentrated under reduced pressure. The resultingresidue was purified by column chromatography (silica gel, methylenechloride/methanol=20/1→10/1), and the resulting orange solid was washedwith methanol to obtain the title compound(E)-3-[8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (742 mg, 54%) as pale yellow solid.

m.p.: 254-257° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.32 (9H, s), 1.46 (9H, s), 6.85 (1H, s), (6.87 (1H,d, J=15.7Hz), 7.82 (1H, d, J=15.9Hz), 7.90-7.93 (2H, m), 9.09 (1H, d,J=7.10Hz)

LRMS-FAB; m/z: 471 (MH⁺)

IR (cm⁻¹): 1678, 1603, 1513, 1308, 1349, 1250, 1146, 727, 440

(B)(E)-3-{2-[4-(2-t-Butoxycarbonylamino-5-[(t-butoxycarbonyl)amino]-{[(t-butoxycarbonyl)imino]methyl}aminopentanoyl)piperazin-1-yl]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester

A solution of(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin3-yl]-2-propenoicacid t-butyl ester (117 mg, 0.957 mmol) in N,N-dimethylformamide (100ml) was added with a solution of tosyl chloride (182 mg, 0.957 mmol) inN,N-dimethylformamide (5.0 ml) and stirred at room temperature for 3hours. This reaction mixture was added with piperazine (275 mg, 3.19mmol) and further stirred overnight at room temperature. The reactionmixture was concentrated under reduced pressure to obtain an orangeresidue.

This residue and Boc-Arg(Boc)₂OH (908 mg, 1.91 mmol) were dissolved in amixed solvent of N,N-dimethylformamide (15 ml) and methylene chloride(25 ml), added with EDC.HCl (489 mg, 2.55 mmol) with ice cooling andstirred overnight at room temperature. The reaction mixture wasconcentrated under reduced pressure, and the resulting residue waspurified by column chromatography (silica gel, methylenechloride:methanol=30:1). The resulting crude product was furtherpurified by column chromatography (silica gel, n-hexane:ethylacetate=2:1) to obtain the title compound(E)-3-{2-[4-(2-t-butoxycarbonylamino-5[(t-butoxycarbonyl)amino]{[(t-butoxycarbonyl)imino]methyl}aminopentanoyl)piperazin-1-yl]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid t-butyl ester (294 mg, 46%) as yellow amorphous solid.

¹H-NMR (CDCl₃) δ: 1.26-1.71 (49H, m), 3.50-3.94 (10H, m), 4.63 (1H,brs), 5.59 (1H, d, J=8.33Hz), 6.61 (1H, a), 7.11 (1H, d, J=15.4Hz),7.44-7.51 (2H, m), 7.97 (1H, s), 9.03 (1H, d, J=7.35Hz)

(C)(E)-3-[2-[4-((2S)-2-Amino-5-{([amino(imino)methyl]amino}pentanoyl)piperazino]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

A solution of(E)-3-(2-[4-(2-t-butoxycarbonylamino5-[(t-butoxycarbonyl)amino]{[(t-butoxycarbonyl)-imino]methyl}aminopentanoyl)piperazin-1-yl]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester (294 mg, 0.296 mmol) in trifluoroacetic acid (30 ml)was stirred at room temperature for 2 hours, and the reaction mixturewas concentrated under reduced pressure. The mixture was subjected toazeotropy with toluene three times, and the resulting residue was washedwith diethyl ether. Further, a procedure in which the resulting residuewas added with formic acid and concentrated under reduced pressure wasrepeated three times, and the residue was washed with diethyl ether toobtain the title compound (E)3-[2-[4-((2S)-2-amino-5-{[amino(imino)methyl]amino}pentanoyl)piperazino]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid (191 mg, 86%) as orange powder.

m.p.: 240° C. (decomp.)

¹H-NMR (CD₃OD) δ: 1.35 (9H, a), 1.61-1.86 (4H, m), 3.25-3.88 (10H, m),4.34 (1H, brs), 6.70 (1H, a), 7.08 (1H, d, J=16.1Hz), 7.45 (1H, d,J=15.6Hz), 7.59-7.67 (1H, m), 7.91 (1H, s), 8.48 (2H, s), 8.91 (1H, d,J=7.32Hz)

LRMS-FAB; m/z: 639 (MH⁺)

IR (cm⁻¹): 1631, 1514, 1435, 1365, 1225, 702, 646

Anal.(for C₂₉H₃₈N₁₀O₅S.2.0 formic acid.1.0 H₂O): Calcd.: C, 49.72; H,5.92; N, 18.71. Found: C, 50.19; H, 6.35;.N, 18.24.

Example 195(E)-3-[2-[4-(5-Aminopentanoyl)piperazino]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A)(E)-3-[2-(4-5-(t-Butoxycarbonyl)aminopentanoylpiperazino)-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester

A solution of(E)-3-[8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin3-yl]-2-propenoicacid t-butyl ester (50.0 mg, 0.106 mmol) in N,N-dimethylformamide (15ml) was added with 4-dimethylaminopyridine (19.4 mg, 0.159 mmol) andslowly added dropwise with a solution of tosyl chloride (30.3 mg, 0.159mmol) in N,N-dimethylformamide (3.0 ml) at room temperature. Thereaction mixture was stirred at room temperature for 4 hours and 30minutes, added with piperazine (45.7 mg, 0.530 mmol) and further stirredovernight at room temperature. The reaction mixture was concentratedunder reduced pressure, and then the resulting residue was dissolved ina mixture solution of N,N-dimethylformamide (3.0 ml) and methylenechloride (10 ml) and added with 5-(t-butoxycarbonylamino)valeric acid(46.1 mg, 0.212 mmol). The mixture was added with EDC.HCl (61.0 mg,0.318 mmol) with ice cooling and the reaction mixture was stirredovernight at room temperature. The reaction mixture was concentratedunder reduced pressure, and the resulting residue was purified by thinlayer chromatography (silica gel, methylene chloride:methanol=30:1,developed twice) to obtain the title compound (E) 3-[2-(4-5-┌(t-butoxycarbonyl)amino┘pentanoylpiperazino)-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (31.3 mg, 41%) as yellow amorphous solid.

¹H-NMR (CD₃OD) δ: 1.26-1.71 (31H, m), 2.48 (2H, t, J=7.47Hz), 3.05-3.08(2H, m), 3.64-3.76 (8H, m), 6.73 (1H, s), 7.00 (1H, dd, J=4.65, 15.7Hz),7.51 (1H, dd, J=4.41, 15.7Hz), 7.61-7.66 (1H, m), 8.02 (1H, s), 8.96(1H, t, J=6.86Hz)

(B)(E)-3-[2-[4-(5-Aminopentanoyl)piperazino]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoic acid

((E)-3-[2-(4-5-┌(t-Butoxycarbonyl)amino┘pentanoylpiperazino)-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (31.3 mg, 0.0432 mmol) was dissolved intrifluoroacetic acid (5.0 ml) and stirred at room temperature for 1hour. The reaction mixture was concentrated, and the resulting residuewas purified by high performance liquid chromatography (MeCN/H₂O system,containing 0.1% formic acid). The resulting amorphous solid was washedwith methanol and diethyl ether to obtain the title compound(E)-3-[2-[4-(5-aminopentanoyl)piperazino]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid (14 mg, 48%) as yellow powder.

m.p.: 194-198° C.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.72 (4H, brs), 2.53 (2H, brs), 2.96(2H, brs), 3.64-3.76 (8H, m), 6.73 (1H, s), 7.06 (1H, d, J=15.4Hz), 7.56(1H, d, J=15.6Hz), 7.61 (1H, d, J=7.57Hz), 7.99 (1H, s), 8.43 (1H, s),8.95 (1H, d, J=7.32Hz)

LRMS-FAB; m/z: 582 (MH⁺)

IR (cm⁻¹): 2964, 1666, 1633, 1599, 1514, 1435, 1365, 1319, 1201, 1132,1014, 985, 741, 702

Anal. (for C₂₈H₃N₇O₅S.2.0 formic acid.1.75H₂O): Calcd.: C, 51.09; H,6.07; N, 13.90. Found: C, 50.75; H, 5.70; N, 13.86.

Example 196(E)-3-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-{4-[5-(2-pyrimidinylamino)pentanoyl]piperazino}-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A) 2-(5-Hydroxypentyl)-1,3-isoindolinedione

A solution of 3-amino]-pentanol (25.0 g, 242 mmol) and1,3-dihydro-1,3-isobenzofurandione (35.9 g, 242 mmol) in toluene (300ml) was refluxed overnight by heating. The reaction mixture wasconcentrated under reduced pressure, and the residue was dissolved inmethylene chloride, washed with 1 N hydrochloric acid, saturated aqueoussodium hydrogencarbonate and saturated brine and further dried overanhydrous sodium sulfate. After filtration, the solvent was evaporatedunder reduced pressure to obtain the title compound2-(5-hydroxypentyl)-1,3-isoindolinedione (55.5 g, 98%) as pale yellowsyrup.

¹H-NMR (CDCl₃) δ: 1.38-1.46 (2H, m), 1.59-1.76 (4H, m), 3.64-3.72 (4H,m), 7.69-7.72 (2H, m), 7.82-7.85 (2H, m)

(B) 5-(1,3-Dioxo-2,3-dihydro-1H-2-isoindolyl)pentanoic acid A mixedsolution of 2-(5-hydroxypentyl)-1,3-isoindolinedione (10.0 g, 42.9 mmol)and sodium periodate (36.7 g, 171.6 mmol) in carbon tetrachloride (140ml), acetonitrile (140 ml) and water (220 ml) was added with rutheniumchloride n-hydrate (178 mg, 0.858 mmol) with ice cooling, and thereaction mixture was stirred for 3 hours while maintained at 5-18° C.The organic layer was separated, and the aqueous layer was extractedtwice with methylene chloride. The organic layers were combined andwashed with saturated brine. The organic layer was dried over anhydroussodium sulfate, and the solvent was evaporated under reduced pressure toobtain the title compound5-(1,3-dioxo-2,3-dihydro-1H-2-isoindolyl)pentanoic acid (10.7 g, 100%)as gray solid.

¹H-NMR (CDCl₃) δ: 1.65-1.79 (4H, m), 2.41 (2H, t, J=7.20Hz), 3.72 (2H,t, 6.71Hz), 7.70-7.72 (2H, m), 7.83-7.85 (2H, m)

(C) 5-(1,3-Dioxo-2,3-dihydro-1H-2-isoindolyl)pentanoic acid t-butylester

A solution of 5-(1,3-dioxo-2,3-dihydro-1H-2-isoindolyl)pentanoic acid(10.7 g, 43.3 mmol) and 4-dimethylaminopyridine (529 mg, 4.33 mmol) int-butyl alcohol (100 ml) was added with a solution of di-t-butyldicarbonate (14.2 g, 65.0 mmol) in t-butyl alcohol (20 ml) and stirredat 60° C. for 1 hour. The reaction mixture was concentrated underreduced pressure, and the residue was dissolved in methylene chloride,washed with 1 N hydrochloric acid, saturated aqueous sodiumhydrogencarbonate and saturated brine and further dried over anhydroussodium sulfate. After filtration, the solvent was evaporated underreduced pressure to obtain the title compound5-(1,3-dioxo-2,3-dihydro-1H-2-isoindolyl)pentanoic acid t-butyl ester(6.48 g, 49%) as colorless syrup.

¹H-NMR (CDCl₃) δ: 1.43 (9H, s), 1.59-1.76 (4H, m), 2.26 (2H, t,J=7.23Hz), 3.70 (2H, t, J=6.89Hz), 7.68-7.73 (2H, m), 7.81-7.86 (2H, m)

(D) 5-Aminopentanoic acid t-butyl ester

A solution of 5-(1,3-dioxo-2,3-dihydro-1H-2-isoindolyl)pentanoic acidt-butyl ester (6.48 g, 21.4 mmol) in ethanol (500 ml) was added withhydrazine monohydrate and stirred at room temperature for 18 hours. Theproduced white precipitates were removed to obtain the title compound5-aminopentanoic acid t-butyl ester (3.30 g, 82%) as pale yellow oilysubstance.

¹H-NMR (CDCl₃) δ: 1.41-1.50 (11H, m), 1.58-1.66 (2H, m), 2.23 (2H, t,7.46Hz), 2.70 (2H, t, J=6.97Hz)

(E) 5-(2-Pyrimidinylamino)pentanoic acid t-butyl ester

A solution of 5-aminopentanoic acid t-butyl ester (100 mg, 0.577 mmol)and ethyldiisopropylamine (224 mg, 1.73 mmol) in dimethyl sulfoxide (10ml) was added with 2-bromopyrimidine (91.7 mg, 0.577 mmol) and stirredovernight at 120° C. The reaction mixture was added with water andextracted with methylene chloride (three times), and the organic layerwas washed with saturated brine and dried over anhydrous sodium sulfate.The solvent was evaporated under reduced pressure, and the crude productwas purified by thin layer chromatography (methylenechloride:methanol=9:1) to obtain the title compound5-(2-pyrimidinylamino) pentanoic acid t-butyl ester (92.9 mg, 64%) aspale yellow oily substance.

¹H-NMR (CDCl₃) δ: 1.44 (9H, 9), 1.59-1.73 (4H, m), 2.27 (2H, t,J=7.09Hz), 3.38-3.44 (2H, m), 5.22 (1H, brs), 6.50 (1H, t, J=4.77Hz),8.26 (2H, d, J=4.65Hz)

(F) 5-(2-Pyrimidinylamino)pentanoic acid trifluoroacetate

A solution of 5-(2-pyrimidinylamino)pentanoic acid t-butyl ester (92.9mg, 0.370 mmol) in methylene chloride (5.0 ml) was added withtrifluoroacetic acid (5.0 ml) and stirred at room temperature for 1hour. The reaction mixture was concentrated under reduced pressure toobtain the title compound 5-(2-pyrimidinylamino)pentanoic acidtrifluoroacetate (122 mg, >100%) as a brown amorphous solid.

¹H-NMR (CD₃OD) δ: 1.68 (4H, brs), 2.33-2.36 (2H, m), 3.46-3.49 (2H, m),6.86 (1H, t, J=5.27Hz), 8.47 (2H, brs).

(G)(E)-3-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-{4-[5-(2-pyrimidinylamino)pentanol]-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester

A solution of(E)-3-[8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (100 mg, 0.213 mmol) in dimethylformamide (30 ml) wassuccessively added with 4-dimethylaminopyridine (39.1 mg, 0.320 mmol)and p-toluenesulfonyl chloride (61.0 mg, 0.320 mmol) and stirred at roomtemperature for 4 hours. The reaction mixture was further added withpiperazine (92.2 mg, 1.07 mmol) and stirred overnight, and the solventwas evaporated under reduced pressure to obtain an orange residue.

The residue was dissolved in dimethylformamide (10 ml)/methylenechloride (30 ml), added with 5-(2-pyrimidinylamino)pentanoic acidtrifluoroacetate (123 mg, 0.398 mmol), triethylamine (59.1 μl, 0.426mmol) and further EDC.HCl (123 mg, 0.639 mmol), and stirred overnight atroom temperature. The reaction mixture was concentrated, and the residuewas purified by thin layer chromatography (methylenechloride:methanol=9:1). The target fraction was extracted with methylenechloride/methanol (9:1), and further the resulting crude product waspurified by thin layer chromatography (ethyl acetate) again to obtainthe title compound(E)-3-(8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl-4-oxo-2-{4-[5-(2-pyrimidinylamino)pentanol]-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester (46.3 mg, 30%) as yellow amorphous solid.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.53 (9H, s), 1.56-1.81 (4H, m),2.41-2.45 (2H, m), 3.42-3.76 (10H, m), 5.30 (1H, s), 6.51 (1H, t,J=4.77Hz), 6.60 (1H, s), 7.11 (1H, d, J=15.7Hz), 7.44-7.50 (2H, m), 7.93(1H, d, J=1.22Hz), 8.26 (2H, d, J=4.65Hz), 9.00 (1H, d, J=7.58Hz)

ESI-MS; m/z: 717 (MH⁺)

(H)(E)-3-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-{4-[5-(2-pyrimidinylamino)pentanoyl]piperazino}-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid 1.0 trifluoroacetate

A solution of(E)-3-(8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-{4-[5-(2-pyrimidinylamino)pentanol]-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester (46.3 mg, 0.0647 mmol) dissolved in methylenechloride (10 ml) was added with trifluoroacetic acid (10 ml) and stirredat room temperature for 1 hour and 30 minutes. The reaction mixture wasconcentrated under reduced pressure, and the resulting solid was washedwith diethyl ether to obtain the title compound(E)-3-(8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-{4-[5-(2-pyrimidinylamino)pentanoyl]piperazino}-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid 1.0 trifluoroacetate (32.5 mg, 65%).

m.p. 234-238° C.

Anal. (for C₃₃H₃₇N₉O₅S.1.0TFA): Calcd.: C, 52.78; H, 4.95; N, 16.29.Found: C, 52.65; H, 5.19; N, 16.10.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.56 (414, brs), 2.39 (2H, brs),3.28-3.62 (10H, m), 6.57 (1H, t, J=4.88Hz), 6.86 (1H, s), 6.96 (1H, d,J=15.6Hz), 7.38 (1H, brs), 7.47 (1H, d, J=15.6Hz), 7.66 (1H, d,J=7.32Hz), 8.22 (1H, s), 8.29 (2H, d, J=7.32Hz)

FAB-MS; m/z: 660 (MH⁺)

IR (cm⁻¹): 1672, 1518, 1442, 1286, 1200, 1140, 984, 721

Example 197(E)-3-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-{4-[5-(1,4,5,6-tetrahydro-2-pyrimidinylamino)pentanoyl]piperazino}-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A) 2-(Methylsulfanyl)-1,4,5,6-tetrahydropyrimidine hydroiodide

3,4,5,6-Tetrahydro-2-pyrimidinethiol (5.0 g, 43.0 mmol) was dissolved inacetone (60 ml), added with methyl iodide (2.68 ml, 43.0 mmol) andrefluxed by heating for 10 minutes. This reaction solution was addedwith ethanol and n-hexane and cooled with ice. The deposited crystalswere collected by filtration to obtain the title compound (10.8 g) aswhite powder.

¹H-NMR (CD₃OD) δ: 2.02-2.08 (2H, m), 2.63 (3H, d, J=3.42Hz), 3.48-3.52(4H, m)

(B) 5-(1,4,5,6-Tetrahydro-2-pyrimidinylamino)pentanoic acid benzyl ester

2-(Methylsulfanyl)-1,4,5,6-tetrahydropyrimidine hydroiodide (745 mg,2.89 mmol), 5-aminopentanoic acid t-butyl ester (500 mg, 2.89 mmol) andtriethylamine (400 μl) were dissolved in dimethylformamide (5.0 ml) andstirred at 100° C. for 26 hours. The solution was cooled to roomtemperature and concentrated under reduced pressure, and then theresidue was purified by column chromatography (silica gel, chloroform,chloroform:acetone=9:1, chloroform:methanol=9:1) to obtain the titlecompound (410 mg) as pale yellow oily substance.

¹H-NMR (CDCl₃) δ: 1.45 (9H, s), 1.65-1.68 (4H, m), 1.96-2.02 (2H, m),2.30 (2H, t, J=6.59Hz), 3.17-3.22 (2H, m), 3.40-3.43 (4H, m), 7.13 (1H,brs), 7.63 (1H, brs)

(C) 5-(1,4,5,6-Tetrahydro-2-pyrimidinylamino)pentanoic acid

5-(1,4,5,6-Tetrahydro-2-pyrimidinylamino)pentanoic acid benzyl ester(109 mg, 0.426 mmol) was dissolved in methylene chloride (5.0 ml), addedwith trifluoroacetic acid (5.0 ml) and stirred at room temperature for 1hour 30 minutes. The reaction solution was concentrated under reducedpressure to obtain the title compound (85 mg) as yellow oily substance.

¹H-NMR (CD₃OD) δ: 1.56-1.69 (4H, m), 1.82-1.97 (2H, m), 2.34 (2H, t,J=6.97Hz), 3.12 (2H, t, J=6.72Hz), 3.30-3.36 (6H, m)

(D)(E)-3-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-{4-[5-(1,4,5,6-tetrahydro-2-pyrimidinylamino)pentanoyl]piperazino}-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid t-butyl ester

(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin3-yl]-2-propenoicacid t-butyl ester (100 mg, 0.213 mmol), 4-dimethylaminopyridine (39.1mg, 0.320 mmol) and tosyl chloride (61.0 mg, 0.320 mmol) were dissolvedin dimethylformamide (30 ml) and stirred at room temperature for 3hours. This solution was added with piperazine (92.2 mg,-1.07 mmol),stirred overnight at room temperature and concentrated under reducedpressure.

The resulting residue and5-(1,4,5,6-tetrahydro-2-pyrimidinylamino)valeric acid (85 mg, 0.426mmol) were dissolved in a mixed solution of dimethylformamide (10 ml)and methylene chloride (30 ml), added with triethylamine (59.1 μl, 0.426mmol) and EDC.HCl (123 mg, 0.639 mmol) and stirred at room temperaturefor 2 days. The reaction solution was concentrated under reducedpressure, and the resulting residue was purified by thin layerchromatography (methylene chloride: methanol=9:1, developed twice) toobtain the title compound (32.1 mg) as yellow amorphous solid.

¹H-NMR (CDCl₃) δ: 1.23-1.90 (24H, m), 2.47-2.48 (2H, m), 3.17-3.18 (2H,m), 3.34 (4H, brs), 3.56-3.73 (8H, m), 6.57 (1H, s), 7.08-7.13 (2H, m),7.45 (1H, d, J=15.7Hz), 7.56 (1H, d, J=7.59Hz), 7.74 (1H, d, J=8.08Hz),7.96 (1H, brs), 8.02 (1H, s), 9.00 (1H, d, J=7.35Hz)

LRMS-ESI; m/z: 721 (MH⁺)

(E)(E)-3-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-{4-[5-(1,4,5,6-tetrahydro-2-pyrimidinylamino)pentanoyl]piperazino}-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(E)-3-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-{4-[5-(1,4,5,6-tetrahydro-2-pyrimidinylamino)pentanoyl]piperazino}-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoic acid t-butyl ester (32.1 mg, 0.0446 mmol) was dissolved inmethylene chloride (10 ml), added with trifluoroacetic acid (10 ml) andstirred at room temperature for 3 hours. The reaction solution wasconcentrated under reduced pressure, and then the resulting residue waswashed with diethyl ether and dried under reduced pressure to obtain thetitle compound (26.2 mg) as orange amorphous solid.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.51 (4H, m), 1.81 (2H, t, J=5.13Hz),2.39 (2H, t, J=6.74Hz), 3.06-3.07 (2H, m), 3.24-3.62 (12H, m), 6.86 (1H,s), 6.96 (1H, d, J=15.4Hz), 7.23 (1H, brs), 7.45-7.49 (2H, m), 7.64-7.68(2H, m), 8.19 (1H, s), 8.94 (1H, d, J=7.35Hz)

LRMS-FAB; m/z: 664 (MH⁺)

IR (cm⁻¹): 2968, 1641, 1520, 1439, 1367, 1284, 1173, 1122, 1034, 1011,683, 567

HRMS-FAB (C₃₂H₄₂O₅N₉S); m/z: Calcd. (m/z): 664.3030 Found (m/z):664.3019

Example 198(E)-3-({[4-t-Butyl]-1,3-thiazol-2-yl}amino)carbonyl)-2-{4-[5-(dimethylamino)pentanoyl]piperazino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A) 5-Hydroxypentanoic acid benzyl ester

A solution of o-valerolactone (16.0 g, 160 mmol) in ethanol (160 ml) wasslowly added with a solution of sodium hydroxide (6.72 g, 168 mmol) inwater (42 ml) with ice cooling and stirred overnight at roomtemperature. The reaction mixture was concentrated under reducedpressure, and the residue was subjected to azeotropy with toluene(twice) to removed moisture. The resulting residue was dissolved inN,N-dimethylformamide (200 ml). This solution was added with benzylbromide (19.0 ml, 160 ml) and stirred at room temperature for a wholeday and night. The reaction mixture was concentrated under reducedpressure, and the residue was added with water and extracted twice withethyl acetate. The combined organic layer was washed with water andsaturated brine and further dried over anhydrous sodium sulfate. Aftersodium sulfate was removed by filtration, the filtrate was concentratedunder reduced pressure, and the resulting residue was purified by columnchromatography (silica gel, n-hexane:ethyl acetate=1:1) to obtain thetitle compound 5-hydroxypentanoic acid benzyl ester (18.8 g, 56%) ascolorless syrup.

¹H-NMR (CDCl₃) δ: 1.56-1.78 (4H, m), 2.41 (2H, t, J=7.35Hz), 3.63 (2H,t, J=6.25Hz), 5.12 (2H, s), 7.29-7.38 (5H, m)

LRMS-FAB; m/z: 209 (MH⁺)

IR (ATR) cm⁻¹: 3444, 2939, 1730, 1456, 1151, 1057, 980, 737, 696, 579,496

(B) 5-Bromopentanoic acid benzyl ester

A solution of 5-hydroxypentanoic acid benzyl ester (3.0 g, 14.4 mmol)and triphenylphosphine (4.53 g, 17.3 mmol) in methylene chloride (100ml) was added with carbon tetrabromide (7.16 g, 21.6 mmol) and stirredat room temperature for 30 minutes. The reaction mixture wasconcentrated under reduced pressure, and the resulting residue waspurified by column chromatography (silica gel, n-hexane:ethylacetate=10:1) to obtain the title compound 5-bromopentanoic acid benzylester (2.80 g, 72%) as colorless oily substance.

¹H-NMR (CDCl₃) δ: 1.77-1.94 (4H, m), 2.40 (2H, t, J=7.20Hz), 3.40 (2H,t, J=6.47Hz), 5.12 (2H, s), 7.30-7.40 (5H, m)

LRMS-FAB; m/z: 271 (MH⁺)

IR (ATR) cm⁻¹: 2958, 1732, 1454, 1255, 1163, 737, 696, 561

HRMS-FAB (C₁₂H₁₆O₂Br); m/z: Calcd. (m/z): 271.0334 Found (m/z): 271.0325

(C) 5-(Trimethylamino)pentanoic acid benzyl ester

A solution of 5-bromopentanoic acid benzyl ester (1.00 g, 3.69 mmol) intetrahydrofuran (20 ml) was added with a solution of dimethylamine intetrahydrofuran (2.0 M, 3.69 ml) and stirred at room temperature for 4days. The reaction mixture was concentrated under reduced pressure, andthe resulting residue was dissolved in methylene chloride, washed withsaturated brine and further dried over anhydrous sodium sulfate. Aftersodium sulfate was removed by filtration, the filtrate was concentratedunder reduced pressure, and the resulting residue was purified by columnchromatography (silica gel, methylene chloride:methanol=5:1) to obtainthe title compound 5-(trimethylamino)pentanoic acid benzyl ester (672mg, 77%).

¹H-NMR (CDCl₃) δ: 1.43-1.56 (2H, m), 1.64-1.71 (2H, m), 2.23 (6H, s),2.30 (2H, t, J=7.45Hz), 2.39 (2H, t, J=7.32Hz), 5.12 (2H, s), 7.30-7.39(5H, m)

(D) 5-(Dimethylamino)pentanoic acid

5-(Trimethylamino)pentanoic acid benzyl ester (672 mg, 2.86 mmol) inmethanol (30 ml) was added with a 10% palladium carbon catalyst (M)(containing water, 200 mg) and stirred overnight under hydrogenatmosphere at room temperature. The catalyst was removed by filtration,and then the filtrate was concentrated under reduced pressure to obtainthe title compound 5-(dimethylamino)pentanoic acid (432 mg, 100%) ascolorless syrup.

¹H-NMR (DMSO-d₆) δ: 1.46-1.51 (4H, m), 2.15-2.21 (2H, m), 2.32 (6H, s),2.47-2.52 (2H, m)

(E)(E)-3-({[4-t-Butyl]-1,3-thiazol-2-yl}amino)carbonyl)-2-{4-[5-(dimethylamino)pentanoyl]piperazino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid t-butyl ester

A solution of(E)-3-[8-(4-t-butylthiazol-2-ylcarbamoyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]propenoicacid t-butyl ester (90.6 mg, 0.193 mmol) in N,N-dimethylformamide (30ml) was added with 4-dimethylaminopyridine (35.4 mg, 0.290 mmol) andtosyl chloride (55.3 mg, 0.290 mmol) and stirred overnight at roomtemperature. The reaction mixture was concentrated under reducedpressure, and the resulting residue and 5-(dimethylamino)pentanoic acid(70.1 mg, 0.483 mmol) were dissolved in methylene chloride (30 ml) andN,N-dimethylformamide (10 ml). This mixture was added with EDC.HCl (111mg, 0.579 mmol), stirred overnight at room temperature and concentratedunder reduced pressure. The resulting residue was purified by thin layerchromatography (methylene chloride:methanol=9:1, developed twice), andthe target fraction was extracted with methylene chloride/methanol (9:1)to obtain the title compound(E)-3-{8-(4-t-butylthiazol-2-ylcarbamoyl)-2-[4-(5-dimethylaminopentanoyl)piperazine-1-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}propenoicacid t-butyl ester (70.8 mg, 55%) as orange solid.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.53 (9H, s), 1.72-1.74 (4H, m), 2.44(2H, t, J=6.59Hz), 2.64 (6H, s), 2.79-2.82 (2H, m), 3.51-3.73 (8H, m),6.59 (1H, s), 7.10 (1H, d, J=15.9Hz), 7.46 (1H, d, J=15.6Hz), 7.52 (1H,d, J=7.32Hz), 7.75 (1H, d, J=8.06Hz), 7.96 (1H, s), 8.99 (1H, d,J=7.32Hz)

LRMS-ESI; m/z: 666 (MH⁺)

(F)(E)-3-({[4-t-Butyl]-1,3-thiazol-2-yl}amino)carbonyl)-2-{4-[5-(dimethylamino)pentanoyl]piperazino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

A solution of(E)-3-({[4-t-butyl]-1,3-thiazol-2-yl}amino)carbonyl)-2-{4-[5-(dimethylamino)pentanoyl]piperazino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid t-butyl ester (70.8 mg, 0.106 mmol) in methylene chloride (10 ml)was added with trifluoroacetic acid (10 ml) and stirred for 2 hours atroom temperature. The reaction mixture was concentrated under reducedpressure, and the resulting residue was purified by high performanceliquid chromatography (MeCN/H₂O system, containing 0.1% formic acid).The resulting residue was lyophilized to obtain the title compound (33.3mg) as yellow amorphous solid.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.69-1.78 (4H, m), 2.54 (2H, t,J=6.59Hz), 2.87 (6H, s), 3.11-3.15 (2H, m), 3.63-3.78 (8H, m), 6.74 (1H,s), 7.08 (1H, d, J=15.4Hz), 7.57 (1H, d, J=15.6Hz), 7.63 (1H, d,J=7.57Hz), 8.01 (1H, s), 8.97 (1H, d, J=7.33Hz)

LRMS-FAB; m/z: 610 (MH⁺)

IR (ATR) cm⁻¹: 1637, 1516, 1435, 1365, 1290, 1225, 1011, 874, 683, 567

HRMS-FAB (C₃₀H₄₀O₅N₇S); m/z: Calcd. (m/z): 610.2812 Found (m/z):610.2789

Example 199[5-(4-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-carboxy-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}piperazino)-5-oxopentyl]-(trimethyl)ammoniumiodide

(E)-3-({[4-t-Butyl]-1,3-thiazol-2-yl}amino)carbonyl)-2-{4-[5-(dimethylamino)pentanoyl]piperazino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid (15 mg, 0.0246 mmol) was dissolved in dimethylformamide (10 ml),added with methyl iodide (15 μl, 0.246 mmol) and left overnight in arefrigerator (3° C.). The reaction solution was concentrated underreduced pressure, and the resulting residue was washed with diethylether and dried under reduced pressure to obtain the title compound(15.5 mg) as orange powder.

m.p.: 176-182° C.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.69 (2H, brs), 1.86 (2H, brs), 2.57(2H, t, J=6.96Hz), 2.88-2.90 (2H, m), 3.14 (9H, s), 3.49-3.77 (8H, m),6.76 (1H, s), 7.09 (1H, d, J=15.4Hz), 7.23 (1H, d, J=7.57Hz), 7.62-7.71(2H, m), 8.05 (1H, s), 9.01 (1H, d, J=7.32Hz)

HRMS-FAB (C₃₁H₄₂O₅N₇S); m/z: Calcd. (m/z): 624.2968 Found(m/z):.624.2982

IR (ATR) cm⁻¹: 2956, 1670, 1637, 1597, 1519, 1439, 1225, 1011, 744, 683,631

Example 200(E)-3-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[(hydroxysulfonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A)(E)-3-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[(hydroxysulfonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid t-butyl ester

(E)-3-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester (166 mg, 0.299 mmol) and dicyclohexylcarbodiimide(309 mg, 1.50 mmol) were dissolved in dimethylformamide (11 ml) andadded with a solution of concentrated sulfuric acid (44.0 mg, 0.449mmol) in dimethylformamide (2.0 ml) with ice cooling. The reactionsolution was stirred for 20 minutes and added with triethylamine (2.0ml). The produced precipitates were removed, and the reaction solutionwas concentrated under reduced pressure. The resulting residue waspurified by thin layer chromatography (methylene chloride:methanol=10:1)and then purified by thin layer chromatography (ethylacetate:methanol=9:1, developed twice) again to obtain the titlecompound (72.5 mg) as orange amorphous solid.

¹H-NMR (CD₃OD) δ: 1.34 (9H, s), 1.53 (9H, s), 1.69-1.75 (1H, m),1.84-1.89 (1H, m), 1.98 (1H, brs), 2.15 (1H, brs), 3.46-3.66 (3H, m),4.27 (1H, dd, J=3.29, 13.0Hz), 4.51-4.55 (1H, m), 6.73 (1H, s), 6.95(1H, d, J=15.6Hz), 7.49 (1H, d, J=15.6Hz), 7.54 (1H, d, J=7.31Hz), 8.05(1H, s), 8.92 (1H, d, J=7.43Hz)

LRMS-ESI; m/z: 634 (MH⁺)

(B)(E)-3-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[(hydroxysulfonyl)-oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(E)-3-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[(hydroxysulfonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid t-butyl ester (72.5 mg, 0.114 mmol) was dissolved in methylenechloride (10 ml), added with trifluoroacetic acid (10 ml) and stirred atroom temperature for 4 hours. The reaction solution was concentratedunder reduced pressure, and then the resulting residue was washed withdiethyl ether and methanol and dried under reduced pressure to obtainthe title compound (60 mg) as orange powder.

m.p.: 196° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.65 (2H, m), 1.87 (1H, brs), 2.01(1H, brs), 3.17-3.48 (3H, m), 4.09-4.17 (2H, m), 6.84 (1H, s), 6.92 (1H,d, J=15.4Hz), 7.42 (1H, d, J=15.4Hz), 7.58 (1H, dd, J=1.71, 7.39Hz),8.20 (1H, s), 8.89 (1H, d, J=7.35Hz)

LRMS-FAB; m/z: 578 (MH⁺)

IR (ATR) cm⁻¹: 1684, 1591, 1522, 1437, 1236, 1184, 1005, 960, 862, 798,746, 582

Anal. (as C₂₄H₂₇N₅O₈S₂.0.25TFA.1.75H₂O) Calcd.: C, 46.15; H, 4.86; N,10.98. Found: C, 46.45; H, 4.96; N, 10.74.

Example 201(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-{5-[(hydroxysulfonyl)oxylpentanoyl}piperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A) 5-[1-(t-Butyl)-1,1-diphenylsilyl]oxypentanoic acid benzyl ester

A solution of 5-hydroxypentanoic acid benzyl ester (2.0 g, 9.60 mmol)and imidazole (1.96 g, 28.8 mmol) in dimethylformamide (10 ml) was addedwith a solution of t-butyldiphenylsilyl chloride (3.17 g, 11.5 mmol) indimethylformamide (10 ml) and stirred overnight at room temperature. Thereaction mixture was concentrated under reduced pressure, added withethyl acetate and water and separated, and the organic layer was washedwith water (three times) and saturated brine and dried over anhydroussodium sulfate. The solvent was evaporated under reduced pressure, andthen the resulting residue was purified by column chromatography (silicagel, n-hexane:ethyl acetate=15:1) to obtain the title compound (3.25 g)as colorless oily substance.

¹H-NMR (CDCl₃) δ: 1.04 (s, 9H), 1.56-1.62 (m, 2H), 1.71-1.79 (m, 2H),2.36 (t, J=7.47Hz, 2H), 3.65 (t, J=6.25Hz, 2H), 5.10 (s, 2H), 7.34-7.43(m, 6H), 7.63-7.66 (m, 4H)

(B) 5-[1-t-Butyl]-1,1-diphenylsilyl]oxypentanoic acid

A solution of 5-[1-(t-butyl)-1,1-diphenylsilyl]oxypentanoic acid benzylester (3.25 g, 7.28 mmol) in tetrahydrofuran (100 ml) was added with asolution of lithium hydroxide 1.0 hydrate (914 mg, 21.8 mmol) in water(100 ml) and stirred at room temperature for 6 hours. The reactionsolution was made acidic by addition of 1 N hydrochloric acid andextracted with methylene chloride three times, and then the combinedorganic layer was washed with saturated brine and dried over anhydroussodium sulfate. The solvent was evaporated under reduced pressure toobtain the title compound (2.50 g) as colorless oily substance.

¹H-NMR (CDCl₃) δ: 1.05 (9H, s), 1.57-1.76 (4H, m), 2.36 (2H, t,J=7.35Hz), 3.66-3.68 (2H, m), 7.35-7.42 (6H, m), 7.64-7.67 (4H, m)

IR (ATR) cm⁻¹: 2931, 2858, 1707, 1427, 1105, 822, 739, 698, 613, 503

LRMS-FAB; m/z: 357 (MH⁺)

HRMS-FAB (C₂₁H₂₉O₃Si); m/z: Calcd. (m/z): 357.1886 Found (m/z): 357.1928

(C)(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester

A mixed solution of dimethylformamide (1.12 ml) and methylene chloride(120 ml) was added with oxalyl chloride (1.26 ml, 14.5 mmol) with icecooling and stirred for 15 minutes. This solution was added withN⁸-[4-(t-butyl)-1,3-thiazol-2-yl]-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxamide(2.00 g, 5.81 mmol), stirred at room temperature for 1 hour and 30minutes and added with saturated aqueous sodium hydrogencarbonate. Thissolution was concentrated under reduced pressure, and then this residuewas added with 1 N aqueous hydrochloric acid. The mixture was adjustedto about pH 4.5 and extracted with chloroform five times, and theorganic layer was concentrated under reduced pressure.

The resulting residue was dissolved in a mixed solution oftetrahydrofuran (1000 ml) and dimethylformamide (50 ml), added with(t-butoxycarbonylmethylene)triphenylphosphorane (3.28 g, 8.72 mmol) andstirred overnight at room temperature. The reaction mixture wasconcentrated under reduced pressure, and the resulting residue waswashed with methanol and dried over under reduced pressure to obtain thetitle compound (1.31 g) as yellow solid.

¹H-NMR (DMSO-d₆) δ: 1.32 (9H, s), 1.47 (9H, s), 6.85-6.90 (2H, m),7.82-7.92 (3H, m), 9.07 (1H, d, J=7.08Hz)

LRMS-FAB; m/z: 470 (MH⁺)

IR (ATR) cm⁻¹: 3435, 2972, 1678, 1585, 1514, 1308, 1250, 1146, 987, 854,727, 538, 438

Anal. (for C₂₃H₂₆N₄O₅S.1.75H₂O) Calcd.: C, 55.02; H, 5.92; N, 11.16.Found: C, 55.51; H, 5.91; N, 10.74.

(D) (E)3-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[4-(5-hydroxypentanoyl)piperazino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid t-butyl ester

A solution of (E)3-[8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (200 mg, 0.425 mmol) in dimethylformamide (30 ml) wasadded with 4-dimethylaminopyridine (78 mg, 0.638 mmol) and tosylchloride (122 mg, 0.638 mmol) and stirred at room temperature for 4hours. This reaction solution was added with piperazine (183 mg, 2.13mmol), stirred overnight at room temperature and concentrated underreduced pressure.

The resulting residue was dissolved in a mixed solution of methylenechloride (30 ml) and dimethylformamide (10 ml), added with5-{[1-(t-butyl)-1,1-diphenylsilyl]oxy}valeric acid (455 mg, 1.28 mmol)and EDC.HCl (326 mg, 1.70 mmol) and stirred overnight at roomtemperature. The reaction mixture was concentrated under reducedpressure, and the resulting residue was purified by thin layerchromatography (silica gel, methylene chloride:methanol=9:1) and thenthin layer chromatography (silica gel, n-hexane:ethyl acetate=2:1) againto obtain yellow amorphous solid.

The obtained solid was dissolved in tetrahydrofuran (30 ml), added withtetra-n-butyl ammonium fluoride THF solution (1.0 M solution, 474 μl,0.474 mmol) and stirred at room temperature for 3 hours and 30 minutes.The reaction mixture was concentrated under reduced pressure, and theresulting residue was purified by column chromatography (silica gel,methylene chloride:methanol=20:1). The resulting crude product waspurified by thin layer chromatography (silica gel, ethylacetate:methanol=9:1), and the resulting solid was washed with diethylether to obtain the title compound (116 mg) as yellow powder.

¹H-NMR (CD₃OD) δ: 1.25-1.70 (22H, m), 2.44-2.51 (2H, m), 3.57-3.76 (10H,m), 6.74 (1H, s), 7.01 (1H, d, J=15.6Hz), 7.52 (1H, d, J=15.6Hz), 7.64(1H, m), 8.03 (1H, s), 8.97 (1H, d, J=7.32Hz)

LRMS-FAB; m/z: 639 (MH⁺)

HRMS-FAB (C₃₂H₄₃O₆N₆S); m/z: Calcd. (m/z): 639.2965 Found (m/z):639.2975

(E)(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-{5-[(hydroxysulfonyl)oxy]pentanoyl}piperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester

A solution of(E)-3-{8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[4-(5-hydroxypentanoyl)piperazino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester (101 mg, 0.159 mmol) in dimethylformamide (9 ml) wasadded with dicyclohexylcarbodiimide (164 mg, 0.795 mmol) and added witha solution of concentrated sulfuric acid (12.7 μl, 0.239 mmol) indimethylformamide (1.0 ml) with ice cooling. The reaction solution wasstirred for 40 minutes, then added with triethylamine (2.0 ml) andconcentrated under reduced pressure, and the resulting residue waspurified by thin layer chromatography (silica gel, methylenechloride:methanol=9:1). The resulting solid was purified by thin layerchromatography (silica gel, separated with ethyl acetate:methanol=9:1,then separated with acetate:methanol=5:1). The resulting solid waswashed with diethyl ether and dried under reduced pressure to obtain thetitle compound (60.8 mg) as orange amorphous solid.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.53 (9H,: s), 1.75-1.77 (4H, m), 2.53(2H, t, J=7.32Hz), 3.59-3.76 (8H, m), 4.04 (2H, t, J=5.98Hz), 6.74 (1H,s), 7.00 (1H, d, J=15.6Hz), 7.53 (1H, d, J=15.6Hz), 7.62 (1H, d,J=6.10Hz), 8.06 (1H, s), 8.97 (1H, d, J=7.32Hz)

LRMS-FAB; m/z; 719 (MH⁺)

HRMS-FAB (C₃₂H₄₃O₉N₆S₂); m/z: Calcd.: 719.2533 Found: 719.2578

(F)(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-{5-[(hydroxysulfonyl)oxy]pentanoyl}piperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

A solution of(E)-3-[8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-{5-[(hydroxysulfonyl)oxy]pentanoyl}piperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (60 mg, 0.0835 mmol) in methylene chloride (10 ml)was added with trifluoroacetic acid (10 ml) and stirred at roomtemperature for 2 hours. The reaction mixture was concentrated underreduced pressure, and the resulting solid was washed with diethyl etherand dried under reduced pressure to obtain the title compound (54.6 mg)as orange powder.

m.p.: 230° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.32 (9H, s), 1.55 (4H, brs), 2.33-2.38 (2H, m),3.58-3.72 (10H, m), 6.85 (1H, s), 6.95 (1H, d, J=15.4Hz), 7.48 (1H, d,J=15.4Hz), 7.63 (1H, d, J=7.34Hz), 8.25 (1H, s), 8.93 (1H, d, J=7.58Hz)

IR (ATR) cm⁻¹: 2962, 1676, 1593, 1518, 1433, 1200, 984, 742, 582

LRMS-FAB; m/z: 663 (MH⁺)

HRMS-FAB (C₂₈H₃₅O₉N₆S₂); m/z: Calcd.: 663.1907 Found: 663.1907

Anal. (for C₂₈H₃₄N₆O₉S₂.1.0TFA.2.0H₂O) Calcd.: C, 44.33; H, 4.84; N,10.34. Found: C, 44.18; H, 5.01; N, 10.36.

Example 202(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-{5-[(carboxymethyl)(methyl)amino]pentanoyl}piperazino)-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl]-2-propenoicacid

(A) 5-[[2-(t-Butoxy)-2-oxoethyl](methyl)amino]pentanoic acid benzylester

A solution of 5-bromopentanoic acid benzyl ester (4.05 g, 14.9 mmol),sarcosine t-butyl ester hydrochloride (5.41 g, 29.8 mmol) andtriethylamine (4.53 g, 44.7 mmol) in methylene chloride (160 ml) wasrefluxed overnight by heating and concentrated under reduced pressure.The resulting residue was dissolved in methylene chloride, successivelywashed with water and saturated brine and dried over anhydrous sodiumsulfate. The solvent was evaporated under reduced pressure, and theresulting residue was purified by column chromatography (silica gel,n-hexane:ethyl acetate=3:1) to obtain the title compound (3.48 g) ascolorless oily substance.

¹H-NMR (CDCl₃) δ: 1.46-1.54 (11H, m), 1.61-1.70 (2H, m), 2.33 (3H, s),2.37-2.40 (2H, m), 2.45-2.49 (2H, m), 3.12 (2H, s), 5.11 (2H, s),7.29-7.38 (5H, m)

LRMS-ESI; m/z: 336 (MH⁺)

(B) 5-[[2-(t-Butoxy)-2-oxoethyl](methyl)amino]pentanoic acid

A suspension of 5-[[2-(t-butoxy)-2-oxoethyl](methyl)amino]pentanoic acidbenzyl ester (500 mg, 1.49 mmol) and 10% palladium carbon catalyst inmethanol (20 ml) was stirred overnight at room temperature underhydrogen atmosphere. The catalyst was removed by filtration, and thenthe filtrate was concentrated under reduced pressure to obtain the titlecompound (353 mg) as colorless oily substance.

¹H-NMR (CDCl₃) δ: 1.46-1.59 (13H, m), 2.28-2.31 (2H, m), 2.46 (3H, s),2.63-2.67 (2H, m), 3.29 (2H, s)

LRMS-FAB; m/z: 246 (MH⁺)

HRMS-FAB (C₁₂H₂₄O₄N); m/z: Calcd.: 246.1705 Found: 246.1697

IR (ATR) cm⁻¹: 1728, 1367, 1221, 1151, 1059, 839, 735

(C)(E)-3-[2-(4-{5-[[2-(t-Butoxy)-2-oxoethyl](methyl)amino]pentanoyl}piperazino)-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester

A solution of(E)-3-[8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl))-2-hydroxy-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (150 mg, 0.319 mmol) in dimethylformamide (30 ml) wasadded with 4-dimethylaminopyridine (58.5 mg, 0.479 mmol) and tosylchloride (91.3 mg, 0.479 mmol) and stirred at room temperature for 4hours. This solution was added with piperazine (137 mg, 1.60 mmol) andfurther stirred overnight at room temperature.

The reaction mixture was concentrated under reduced pressure, and theresulting orange solid was dissolved in a mixed solution ofdimethylformamide (10 ml) and methylene chloride (30 ml). This solutionwas added with 5-[[2-(t-butoxy)-2-oxoethyl](methyl)amino]pentanoic acid(196 mg, 0.798 mmol) and EDC.HCl (183 mg, 0.957 mmol), stirred overnightat room temperature and concentrated under reduced pressure. Theresulting residue was purified by thin layer chromatography (silica gel,methylene chloride:methanol=9:1) to obtain a crude product. This productwas purified by thin layer chromatography (silica gel, ethyl acetate)again to obtain the title compound (107 mg) as orange solid.

¹H-NMR (CD₃OD) δ: 1.28-1.69 (31H, m), 2.34 (3H, s), 2.48-2.54 (4H, m),3.18 (2H, s), 3.63-3.76 (8H, m), 6.73 (1H, s), 6.98 (1H, d, J=15.9Hz),7.49 (1H, d, J=15.9Hz), 7.60 (1H, d, J=7.08Hz), 7.99 (1H, s), 8.93 (1H,d, J=7.08Hz)

LRMS-FAB; m/z: 766 (MH⁺)

HRMS-FAB (C₃₉H₅₆O₇N₇S); m/z: Calcd.: 766.3962 Found: 766.3965

IR (ATR) cm⁻¹: 2968, 1671, 1549, 1516, 1433, 1365, 1292, 1223, 1149,1061, 1016, 984, 854, 733, 702

(D)(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-(5-[(carboxymethyl)(methyl)amino]pentanoyl}piperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

A solution of(E)-3-[8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-(5-[(carboxymethyl)(methyl)amino]pentanoyl]piperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (57.1 mg, 0.0745 mmol) in methylene chloride (10 ml)was added with trifluoroacetic acid (10 ml) and stirred at roomtemperature for 2 hours. The reaction solution was concentrated underreduced pressure, and the residue was subjected to azeotropy withtoluene. The resulting solid was washed with diethyl ether and driedunder reduced pressure to obtain the title compound (49.6 mg) as orangepowder.

m.p.: 169-172° C.

¹H-NMR (CD₃OD) δ: 1.35 (9H, B), 1.69-1.84 (4H, m), 2.56 (2H, t,J=7.08Hz), 2.96 (3H, 8s), 3.24 (2H, brs), 3.67-3.79 (8H, m), 4.08 (2H,brs), 6.75 (1H, S), 7.08 (1H, d, J=15.6Hz), 7.61-7.66 (2H, m), 8.04 (1H,d, J=1.22Hz), 9.00 (1H, d, J=7.32Hz)

LRMS-FAB; m/z: 654 (MH⁺)

HRMS-FAB (C₃₁H₄₀O₇N₇S); m/z: Calcd.: 654.2710 Found: 654.2708

IR (ATR) cm⁻¹: 1672, 1520, 1437, 1284, 1180, 1132, 984, 742, 719

Anal. (for C₃₁H₃₉N₇O₇S.1.5TFA.1.25H₂O) Calcd.: C, 48.20; H, 5.12; N,11.57. Found: C, 48.38; H, 4.95; N, 11.20.

Example 203[5-(4-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-carboxy-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}piperazino)-5-oxopentyl](carboxymethyl)dimethylammonium

A solution of(E)-3-[8-({([4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-{5-[(carboxymethyl)(methyl)amino]pentanoyl}piperazino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (50.0 mg, 0.0653 mmol) in dimethylformamide (10 ml)was added with methyl iodide (162 μl, 2.61 mmol) and left stand at −5°C. for 3 days. The reaction solution was concentrated under reducedpressure, and then the residue was dissolved in methylene chloride (10ml), added with trifluoroacetic acid (10 ml) and stirred at roomtemperature for 2 hours. This solution was concentrated under reducedpressure, and the residue was further subjected to azeotropy withtoluene. Then the residue was purified by high performance liquidchromatography (containing 0.1% formic acid, acetonitrile:water=3:7) andlyophilized to obtain the title compound (33.8 mg) as orange solid.

m.p.: 220-228° C.

¹H-NMR (CD₃OD) δ: 1.35 (9H, a), 1.65-1.72 (2h, m), 1.81-1.89 (2H, m),2.57 (2H, t, J=7.09Hz), 3.29 (6H, s), 3.58-3.78 (10H, m), 4.26 (2H, s),6.75 (1H, s), 7.07 (1H, d, J=15.7Hz), 7.61-7.66 (2H, m), 8.04 (1H, d,J=1.22Hz), 8.98 (1H, d, J=7.34Hz)

LRMS-FAB; m/z: 668 (MH⁺)

HRMS-FAB (C₃₂H₄₂O₇N₇S); m/z: Calcd.: 668.2866 Found: 668.2866

IR (ATR) cm⁻¹: 1631, 1516, 1435, 1379, 1308, 1225, 1103, 1012, 984, 872,735, 702

Anal. (for C₃₂H₄₁N₇O₇S.1.5 formic acid.4.25H₂O) Calcd.: C, 49.41; H,6.62; N, 12.04. Found: C, 49.48; H, 6.36; N, 11.70.

Example 204(2-Amino-2-oxoethyl)(2-{[({(3R)-1-(t-butoxy)-3-oxo-1-propenyl}-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinyl}oxy}carbonyl}amino}ethyl}dimethylammonium

(A)N⁸-[4-(t-Butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

Under argon atmosphere,N⁸-[4-(t-butyl)-1,3-thiazol--2-yl]-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxyamide(5.0 g, 14.5 mmol) in a mixed solvent of dimethylformamide (120 ml) andacetonitrile (60 ml) was added with diphenyl chlorophosphate (6.10 ml,29.4 mmol) and diisopropylethylamine (10 ml) with ice cooling andstirred for 40 minutes. This solution was added with(R)-(+)-3-hydroxypiperidine hydrochloride (3.0 g, 21.8 mmol) anddiisopropylethylamine (5.0 ml) and stirred at 90-100° C. for 2 hours.The reaction solution was cooled to room temperature and concentratedunder reduced pressure, and the residue was dissolved in ethyl acetate,washed with water (three times) and saturated brine and dried overanhydrous sodium sulfate. When the solvent was evaporated under reducedpressure, and the residue was added with methylene chloride and leftstand overnight at room temperature, solid precipitated. This solid wastaken by filtration with washing with methanol to obtain the titlecompound (3.75 g) as pale yellow powder.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.60-1.76 (2H, m), 1.97-1.98 (2H, m),3.61 (3H, brs), 3.96 (2H, brs), 5.72 (1H, s), 6.62 (1H, s), 7.31 (1H, d,J=1.71Hz), 7.79 (1H, d, J=1.47Hz), 8.91 (1H, d, J=7.32Hz)

LRMS-FAB; m/z: 428 (MH⁺)

HRMS-FAB (C₂₁H₂₆O₃N₅S); m/z: Calcd.: 428.1756 Found: 428.1724

IR (ATR) cm⁻¹: 2962, 1660, 1637, 1531, 1496, 1410, 1331, 1223, 1063,856, 758, 490

(B)(E)-3-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-formyloxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester

Under argon atmosphere, dimethylformamide (16 ml) was cooled with ice,added with phosphorus oxychloride (1.58 ml, 17.0 mmol) and stirred atroom temperature for 30 minutes. This solution was added with a solutionofN⁸-[4-(t-butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(3.64 g, 8.51 mmol) in dimethylformamide (16 ml) with ice cooling andstirred for 2 hours. This solution was added with saturated aqueoussodium hydrogencarbonate to terminate the reaction and extracted withchloroform three times. The combined organic layer was washed withsaturated brine and dried over anhydrous sodium sulfate. The solvent wasevaporated under reduced pressure, and then the resulting residue wasdissolved in tetrahydrofuran (100 ml), added with(t-butoxycarbonylmethylene)triphenylphosphorane (9.64 g, 25.6 mmol) andrefluxed by heating for 11 hours. The reaction mixture was cooled toroom temperature and concentrated underreduced pressure, and the residuewas purified by column chromatography (silica gel, n-hexane:ethylacetate=3:1-2:1) to obtain the title compound (2.30 g) as orange solid.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.52 (9H, s), 1.68-2.02 (4H, m),3.56-3.88 (4H, m), 5.16 (1H, brs), 6.62 (1H, s), 7.09 (1H, d, J=15.6Hz),7.45-7.49 (2H, m), 7.90 (1H, d, J=1.22Hz), 8.10 (1H, s), 8.98-9.00 (1H,m)

LRMS-FAB; m/z: 582 (MH⁺)

(C)(E)-3-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester

A solution of(E)-3-{8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-formyloxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester (2.30 g, 3.95 mmol) in methanol (40 ml) was addedwith sodium methoxide (962 mg, 17.8 mmol) with ice cooling and stirredfor 10 minutes. This solution was added with saturated brine andextracted with chloroform, and the aqueous layer was further extractedtwice with a mixed solution of chloroform and methanol (9:1). Thecombined organic layer was dried over anhydrous sodium sulfate. Thesolvent was evaporated under reduced pressure, and the residue waspurified by column chromatography (silica gel, methylenechloride:methanol=9:1) to obtain the title compound (1.91 g) as yellowpowder.

¹H-NMR (CDCl₃) δ: 1.37 (9H, s), 1.40-1.91 (13H, m), 3.58-3.68 (3H, m),3.86 (1H, brs), 4.06 (1H, brs), 6.56 (1H, s), 7.06 (1H, d, J=15.7Hz),7.44-7.48 (2H, m), 7.98 (1H, brs), 8.95 (1H, d, J=7.34Hz)

LRMS-FAB; m/z: 554 (MH⁺)

HRMS-FAB (C₂₈H₃₆O₅N₅S); m/z: Calcd.: 554.2437 Found: 554.2464

(D) (E)-3-{8-(4-t-Butylthiazol-2-ylcarbamoyl)-2-[(3R)-3-(2-dimethylaminoethylcarbamoyloxy)piperidin-1-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester

Under argon atmosphere, a solution of(E)-3-{8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester (1.00 g, 1.81 mmol) and pyridine (227 μl, 2.82 mmol)in tetrahydrofuran (20 ml) was added with 2-chloroethyl isocyanate (311μl, 3.62 mmol) and stirred at 80° C. for 7 hours. The solution was addedwith 2-chloroethyl isocyanate (933 μl, 10.9 mmol) again, stirred for 14hours, further added with 2-chloroethyl isocyanate (622 μl) and stirredfor 3 hours. The reaction mixture was cooled to room temperature, addedwith saturated aqueous sodium hydrogencarbonate and extracted withchloroform three times. The combined organic layer was dried overanhydrous sodium sulfate. The solvent was evaporated under reducedpressure, and the resulting residue was purified by columnchromatography (silica gel, methylene chloride:methanol=40:1) to obtainthe title compound (2.06 g) as crude product of yellow amorphous solid.

This solid (300 mg) was dissolved in dimethylformamide (30 ml), addedwith a solution of dimethylamine in tetrahydrofuran (2.0 M, 23 ml) andsodium iodide (4.0 mg) and stirred overnight at 60° C. The reactionmixture was cooled to room temperature and concentrated under reducedpressure, and the resulting residue was purified by columnchromatography (silica gel, methylene chloride:methanol=9:1) to obtainthe title compound (159 mg) as yellow amorphous solid.

¹H-NMR (CDCl₃) δ: 1.33 (9H, s), 1.52 (9H, s), 165-2.13 (4H, m), 2.24(6H, s), 2.47 (2H, brs), 3.31 (2H, brs), 3.49-3.83 (4H, m), 4.89 (1H,brs), 5.54 (1H, brs), 6.60 (1H, s), 7.10 (1H, d, J=15.7Hz), 7.46 (1H,dd, J=1.84, 7.47Hz), 7.54 (1H, d, J=15.4Hz), 7.93 (1H, d, J=1.23Hz),8.98 (1H, d, J=7.35Hz)

(E)(E)-3-{8-t-Butylthiazol-2-ylcarbamoyl}-2-[(3R)-3-(2-dimethylaminoethylcarbamoyloxy)piperidin-1-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

A solution of(E)-3-{8-(4-t-butylthiazol-2-yl-carbamoyl)-2-[(3R)-3-(2-dimethylaminoethylcarbamoyloxy)piperidin-1-yl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoic acid t-butyl ester (159 mg,0.239 mmol) in methylene chloride (10 ml) was added with trifluoroaceticacid (10 ml) and stirred at room temperature for 30 minutes. Thereaction mixture was concentrated under reduced pressure, and theresulting residue was purified by thin layer chromatography (silica gel,methylene chloride:methanol=9:1, thereafter 5:1). The resulting solidwas washed with diethyl ether to obtain the title compound (118 mg) asorange powder.

¹H-NMR (CDCl₃) δ: 3.34 (9H, s), 1.65-1.72 (2H, m), 1.94-1.97 (1H, m),2.13-2.17 (1H, m), 2.56 (6H, s), 2.64-2.68 (1H, m), 2.85-2.90 (1H, m),2.94-3.00 (1H, m), 3.03-3.12 (1H, m), 3.18-3.21 (1H, m), 3.70-3.73 (1H,m), 3.86 (1H, d, J=13.5Hz), 4.14 (1H, d, J=12.7Hz), 4.98 (1H, brs), 6.17(1H, d, J=6.37Hz), 6.61 (1H, s), 6.97 (1H, d, J=15.4Hz), 7.47 (1H, dd,J=7.84, 7.47Hz), 7.81 (1H, d, J=15.7Hz), 7.87 (1H, s), 9.03 (1H, d,J=7.35Hz)

LRMS-FAB; m/z: 612 (MH⁺)

HRMS-FAB (C₂₉H₃₈O₆N₇S); m/z: Calcd.: 612.2604 Found: 612.2103

(F)(2-Amino-2-oxoethyl)(2-{[({(3R)-1-(t-butoxy)-3-oxo-1-propenyl}-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinyl]oxy}carbonyl)amino}ethyl)dimethylammonium

A solution of(E)-3-{8-t-butylthiazol-2-ylcarbamoyl}-2-[(3R)-3-(2-dimethylaminoethylcarbamoyloxy)piperidin-1-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid (116 mg, 0.189 mmol) in dimethylformamide (20 ml) was added withiodoacetamide (52.5 mg, 0.284 mmol) and stirred overnight at roomtemperature. The reaction mixture was concentrated under reducedpressure, and the residue was purified by thin layer chromatography(silica gel, lower layer of chloroform: methanol:water=7:3:1, developedtwice). The resulting solid was washed with diethyl ether to obtain thetitle compound (107 mg) as orange powder.

m.p.: 191-194° C.

¹H-NMR (CD₃OD) δ: 1.34 (9H, s), 1.70 (1H, brs), 1.88-1.91 (1H, m),1.91-2.01 (2H, m), 3.30-3.34 (10H, m), 3.46-3.74 (3H, m), 3.91-3.94 (2H,m), 4.21 (2H, s), 6.74 (1H, s), 7.10 (1H, d, J=15.7Hz), 7.52-7.59 (2H,m), 8.00 (1H, d, J=1.23Hz), 8.95 (1H, d, J=7.35Hz)

LRMS-FAB; m/z: 669 (MH⁺)

HRMS-FAB (C₃₁H₄₁O₇N₈S); m/z: Calcd.: 669.2819 Found: 669.2801

IR (ATR) cm⁻¹: 1658, 1512, 1431, 1362, 1227, 1101, 862, 737, 700

Anal. (for C₃₁H₄₀N₈O₇S.0.25TFA.3.0H₂O) Calcd.: C, 50.36; H, 6.20; N,14.91. Found: C, 50.68; H, 6.36; N, 14.77.

Example 205 [2-({[((3R)1-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-carboxy-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](dimethyl)[2-(methylamino)-2-oxoethyl]ammonium

A solution of(E)-3-{8-t-butylthiazol-2-ylcarbamoyl}-2-[(3R)-3-(2-dimethylaminoethylcarbamoyloxy)piperidin-1-yl-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid (56.9 mg, 0.0930 mmol) in dimethylformamide (10 ml) was added withN¹-methyl-2-iodoacetamide (27.9 mg, 0.140 mmol) and stirred overnight atroom temperature. The reaction mixture was concentrated under reducedpressure, and the residue was purified by thin layer chromatography(silica gel, chloroform:methanol:water=8:3:0.5, developed twice). Theresulting solid was washed with diethyl ether to obtain the titlecompound (46 mg) as orange powder.

m.p.: 185-190° C.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.70 (1H, brs), 1.88-1.99 (3H, m), 2.78(3H, s), 3.30-3.69 (12H, m), 3.90-3.93 (2H, m), 4.16 (2H, s), 6.74 (1H,s), 7.12 (1H, d, 15.7Hz), 7.50-7.60 (2H, m), 8.00 (1H, d, J=1.96Hz),8.95 (1H, d, J=7.35Hz)

LRMS-FAB; m/z: 683 (MH⁺)

IR (ATR) cm⁻¹: 1662, 1510, 1433, 1360, 1227, 735, 700

Anal. (for C₃₂H₄₂N₈O₇S.0.25TFA.2.0H₂O) Calcd.: C, 52.23; H, 6.24; N,14.99; S, 4.29. Found: C, 52.26; H, 6.06; N, 15.17; S, 4.37.

Example 2061-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-4-piperidylcarbamate

(A)N⁸-[4-(t-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-alpyridopyrimidine-8-carboxyamide

A mixed solution of dimethylformamide (1.67 ml, 21.8 mmol) and methylenechloride (50 ml) was added with oxalyl chloride (1.86 ml, 21.8 mmol)with ice cooling and stirred for 25 minutes. This solution was addedwithN⁸-[4-(t-butyl)-1,3-thiazol-2-yl]-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxamide(3.00 g, 8.71 mmol) and methylene chloride (20 ml), stirred at roomtemperature for 1 hour and added with saturated aqueous sodiumhydrogencarbonate to terminate the reaction. The resulting solution wasadjusted to about pH 3 with 1 N aqueous hydrochloric acid. This solutionwas extracted with chloroform (10 times), and the combined organic layerwas concentrated under reduced pressure.

The resulting residue,2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]acetic acid (3.25 g,13.1 mmol), was dissolved in a mixed solution of piperidine (30 ml) andpyridine (200 ml) and refluxed by heating for 3 hours. The reactionmixture was cooled to room temperature, concentrated under reducedpressure and added with 1 N aqueous hydrochloric acid. The precipitatedsolid was collected by filtration and washed with ethyl acetate. Toremove the remaining piperidine, this solid was suspended in 1 N aqueoushydrochloric acid, stirred, then collected by filtration and washed withethyl acetate to obtain the title compound (2.00 g, 41%) as orangepowder.

¹H-NMR (DMSO-d₆) δ: 1.30 (9H, s), 3.71 (3H, 8), 5.57 (2H, 8), 6.86 (1H,d, J=12.0Hz), 6.92 (1H, d, J=8.56Hz), 7.22 (1H, d, J=8.56Hz), 7.70 (1H,d, J=15.9Hz), 7.93 (1H, d, J=7.34Hz), 7.95 (1H, s), 8.05 (1H, d,J=15.7Hz), 9.14 (1H, d, J=7.09Hz)

(B)1-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4-piperidylcarbamate

N⁸-[4-(t-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(200 mg, 0.358 mmol) in a mixed solvent of dimethylformamide (20 ml) andacetonitrile (10 ml) was added with diphenyl chlorophosphate (148 μl,0.756 mmol) and diisopropylethylamine (245 μl, 1.43 mmol) with icecooling and stirred for 20 minutes. This solution was added with4-hydroxypiperidine (543 mg, 0.537 mmol) and stirred at 80-90° C. for 2hours. The reaction mixture was cooled to room temperature andconcentrated under reduced pressure, and the residue was added withethyl acetate, successively washed three times with water and once withsaturated brine. The solvent was evaporated under reduced pressure, andthe residue was purified by thin layer chromatography (silica gel,chloroform:methanol=9:1) and further purified by thin layerchromatography (silica gel, n-hexane:ethyl acetate=1:3) to obtain thecrude product as orange solid. This solid was dissolved in ethyl acetate(5.0 ml), added with trichloroethyl isocyanate (130 μl, 1.10 mmol) andstirred at room temperature for 85 minutes. This solution was added witha mixed solution of chloroform and methanol (10:1, 10 ml) andconcentrated under reduced pressure. The resulting residue was addedwith tetrahydrofuran (40 ml), water (20 ml) and sodium formate (74.8 mg,1.10 mmol) and stirred at room temperature for 3 hours and 30 minutes.The reaction mixture was concentrated under reduced pressure andextracted with chloroform three times. The combined organic layer waswashed with saturated brine and dried over anhydrous magnesium sulfate.Further, the solvent was evaporated under reduced pressure, and theresulting residue was purified by thin layer chromatography (silica gel,methylene chloride:methanol=9:1) to obtain the title compound (157 mg)as yellow solid.

¹H-NMR (CDCl₃) δ: 1.32 (9H, s), 1.81-2.04 (4H, m),3.08-3.16 (1H, m),3.44-3.59 (3H, m), 3.78-3.82 (1H, m), 4.63-4.77 (1H, m), 4.97-4.99 (1H,m), 5.51 (2H, s), 6.64 (1H, s), 6.87 (1H, d, J=8.82Hz), 7.15-7.18 (1H,m), 7.25-7.36 (3H, m), 7.55 (1H, dd, J=1.84, 7.47Hz), 7.71 (1H, d,J=15.4Hz), 7.88 (1H, d, J=15.7Hz), 8.14 (1H, s), 9.01 (1H, d, J=7.58Hz)

LRMS-ESI; m/z: 685 (MH⁺)

(C)1-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-4-piperidylcarbamate

1-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4-piperidylcarbamate(157 mg, 0.229 mmol) was dissolved in trifluoroacetic acid (30 ml) andstirred at 60° C. for 3 hours. The reaction mixture was cooled to roomtemperature and concentrated under reduced pressure, and the residue waspurified by thin layer chromatography (silica gel, lower layer ofchloroform:methanol:water=7:3:1) to obtain the title compound (21.6 mg)as orange solid.

m.p.: 270-273° C. (decomp.)

¹H-NMR (DMSO-d₆) δ: 1.30 (9H, s), 1.70-1.72 (2H, m), 1.99 (2H, brs),3.32-3.40 (2H, m), 3.86-3.89 (2H, m), 4.77 (1H, brs), 6.51 (2H, brs),6.86 (1H, s), 7.47 (1H, d, J=16.1Hz), 7.63 (1H, d, J=8.54Hz), 7.82 (1H,d, J=15.8Hz), 8.23 (1H, s), 8.94 (1H, d, J=7.32Hz)

LRMS-FAB; m/z: 565 (MH⁺)

IR (ATR) cm⁻¹: 1716, 1637, 1601, 1502, 1442, 1406, 1333, 1227, 1065, 754

Anal. (for C₂₅H₂₈N₁₀O₄S.0.50TFA) Calcd.: C, 50.24; H, 4.62; N, 22.53; S,5.16. Found: C, 50.21; H, 4.77; N, 22.45; S, 5.22.

Example 207(3R)-1-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinylsulfamate

(A) Sulfamoyl chloride

Under argon atmosphere, a solution of chlorosulfonyl isocyanate (3.08ml, 35.3 mmol) in methylene chloride (15 ml) was added with a solutionof formic acid (1.38 ml, 36.4 mmol) in methylene chloride (15 ml) littleby little over 1 hour and 30 minutes. The reaction mixture was stirredovernight, refluxed by heating for 1 hour and further stirred at roomtemperature for 24 hours. This reaction solution was used as 1.18 Mmethylene chloride solution in the following steps (Ref. TetrahedronVol.50, No.23, pp.6825-6838, 1994).

(B)N⁸-[4-(t-Butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxyamide

A solution ofN⁸-[4-(t-butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(1.00 g, 1.79 mmol) in dimethylformamide (50 ml) was added with diphenylchlorophosphate (962 mg, 3.58 mmol) and diisopropylethylamine (1.22 ml,7.16 mmol) with ice cooling and stirred for 40 minutes. This solutionwas added with (R)-(+)-3-hydroxypiperidine hydrochloride (739 mg, 5.37mmol) and diisopropylethylamine (1.84 ml, 10.7 mmol) and stirred at80-90° C. for 2 hours. The reaction solution was cooled to roomtemperature and concentrated under reduced pressure, and the residue waspurified by column chromatography (silica gel, chloroform:ethylacetate=1:1, chloroform:methanol=100:1) to obtain the title compound(793 mg) as orange solid.

¹H-NMR (CDCl₃) δ: 1.38 (9H, s)1.54-1.90 (4H, m), 3.53 (1H, brs),3.64-3.67 (2H, m), 3.78-3.86 (4H, m), 4.08 (1H, brs), 5.52 (2H, s), 6.52(1H, s), 6.87-6.92 (2H, m), 7.14-7.36 (4H, m), 7.46 (1H, d, J=7.32Hz),7.81 (1H, s), 8.00 (1H, brs), 8.88 (1H, d, J=7.57Hz)

(C)(3R)-1-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)hexahydro-3-pyridinylsulfamate

A solution ofN⁸-[4-(t-butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(793 mg, 1.24 mmol) and triethylamine (2.06 ml, 14.9 mmol) in methylenechloride (100 ml) with ice cooling was added with sulfamoyl chloride(1.18 mmol/ml methylene chloride solution, 6.30 ml, 5.33 mmol) and4-dimethylaminopyridine (75.7 mg, 0.620 mmol) and stirred overnight atroom temperature. The reaction solution was concentrated under reducedpressure, added with water and extracted with methylene chloride threetimes. The combined organic layer was concentrated under reducedpressure. When the resulting residue was added with a mixed solution ofn-hexane and ethyl acetate (1:1), solid precipitated. The solid wascollected by filtration to obtain the title compound (404 mg) as yellowpowder.

m.p.: 196-201° C.

¹H-NMR (CDCl₃) δ: 1.37 (9H, s), 2.05 (4H, brs), 3.50-3.79 (7H, m), 4.85(1H, brs), 5.53 (2H, s), 6.59 (1H, s), 6.91 (2H, d, J=8.79Hz), 7.37 (2H,d, J=8.55Hz), 7.45 (1H, brs), 7.73 (1H, d, J=18.6Hz), 7.91 (1H, s), 8.03(1H, d, J=14.9Hz), 8.96 (1H, s)

LRMS-ESI; m/z: 721 (MH⁺)

(D)(3R)-1-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinylsulfamate

(3R)-1-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylsulfamate(840 mg, 1.17 mmol) was dissolved in trifluoroacetic acid (50 ml) andanisole (5.0 ml), stirred overnight at room temperature and then stirredat 50° C. for 7 hours and 30 minutes. The solution was concentratedunder reduced pressure, and the residue was purified by columnchromatography (silica gel, chloroform:methanol=20:1-9:1). The resultingsolid was dissolved in a lower layer of chloroform:methanol:water=7:3:1and filtered through a cotton plug to obtain the title compound (454 mg)as pale yellow powder.

m.p.: 238° C. (decomp.)

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.77 (1H, brs), 1.94-2.03 (2H, m),2.14-2.15 (1H, m), 3.63-3.68 (3H, m), 4.11 (1H, d, J=13.0Hz), 4.73-4.77(1H, m), 6.73 (1H, s), 7.56-7.62 (2H, m), 7.89 (1H, d, J=16.1Hz), 8.04(1H, s), 8.97-8.99 (1H, m)

LRMS-FAB; m/z: 601 (MH⁺)

IR (ATR) cm⁻¹: 2962, 1606, 1496, 1442, 1385, 1333, 1240, 1176, 1099,962, 862, 804, 762, 735, 704, 542

Anal. (for C₂₄H₂₈N₁₁O₅S_(2.)1.0H₂O) Calcd.: C, 46.59; H, 4.89; N, 22.64.Found: C, 46.70; H, 5.11; N, 22.24.

Example 208[2-({[(1-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-4-piperidyl)oxy]carbonyl}amino)ethyl](trimethyl)ammonium

(A)1-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4-piperidyl-N-[2-(dimethylamino)ethyl]carbamate

A solution ofN⁸-[4-(t-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(400 mg, 0.716 mmol) dissolved in a mixture of dimethylformamide (50 ml)and acetonitrile (25 ml) was added with diphenyl chlorophosphate (384mg, 1.43 mmol) and diisopropylethylamine (489 μl, 2.86 mmol) with icecooling and stirred for 40 minutes. This solution was added with4-hydroxypiperidine (108 mg, 1.07 mmol) and stirred at 80-90° C. for 2hours. The reaction solution was cooled to room temperature andconcentrated under reduced pressure, and the reside was purified by thinlayer chromatography (silica gel, methylene chloride:methanol=9:1) toobtain yellow solid.

This solid was dissolved in tetrahydrofuran (10 ml), added with pyridine(39.7 μl, 0.493 mmol) and added with 2-chloroethyl isocyanate (510 μl,5.94 mmol) as 7 divided portions at 80° C. with stirring. After stirredfor 5 days, the solution was added with saturated aqueous sodiumhydrogencarbonate and extracted with chloroform three times. Thecombined organic layer was washed with saturated brine and dried overanhydrous sodium sulfate. The solvent was evaporated under reducedpressure, and the residue was purified by thin layer chromatography(silica gel, methylene chloride:methanol=9:1) to obtain yellow solid.

This solid was dissolved in dimethylformamide (20 ml), added with sodiumiodide (30 mg) and a solution of dimethylamine in tetrahydrofuran (2.0M, 20 ml) and stirred at 60° C. for 3 hours. This solution was furtheradded with sodium iodide (30 mg) and a solution of dimethylamine intetrahydrofuran (2.0 M, 20 ml), stirred for 5 hours and concentratedunder reduced pressure. The resulting residue was purified by thin layerchromatography (silica gel, methylene chloride:methanol=9:1) to obtainthe title compound (184.2 mg) as yellow solid.

LRMS-ESI; m/z: 756 (MH⁺)

(B){2-[({[1-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)-4-piperidyl]oxy}carbonyl)amino]ethyl}(trimethyl)ammonium

1-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-((E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4-piperidyl-N-[2-(dimethylamino)ethyl]carbamate(86.2 mg, 0.114 mmol) was dissolved in dimethylformamide (10 ml), addedwith methyl iodide (328 μl, 5.28 mmol) and left stand overnight at −5°C. The reaction mixture was further added with methyl iodide (656 μl,10.6 mmol) and left stand overnight at −5° C. Then the solution wasconcentrated under reduced pressure, and the residue was purified bythin layer chromatography (silica gel, lower layer ofchloroform:methanol:water=7:3:1) to obtain the title compound (69.3 mg,79%) as orange solid.

LRMS-ESI; m/z: 771 (MH⁺)

(C)[2-({[(1-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-4-piperidyl)oxy]-carbonyl}amino)ethyl](trimethyl)ammonium

{2-[({[1-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4-piperidyl]oxy}carbonyl)amino]ethyl}(trimethyl)ammoniumwas dissolved in trifluoroacetic acid (20 ml) and stirred at 60° C. for4 hours. The reaction mixture was cooled to room temperature andconcentrated under reduced pressure, and the residue was purified bythin layer chromatography (silica gel,chloroform:methanol:water=8:3:0.5). The resulting solid was washed withdiethyl ether to obtain the title compound (44.8 mg, 78%) as orangepowder.

m.p.: 210-215° C.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.86 (2H, brs), 2.06 (2H, brs), 3.19(9H, s), 3.46-3.63 (6H, m), 3.88 (2H, brs), 4.90 (1H, brs), 6.74 (1H,s), 7.39 (1H, d, J=15.6Hz), 7.58 (1H, d, J=7.08Hz), 7.89-8.01 (2H, m),8.98 (1H, d, J=7.33Hz)

LRMS-ESI; m/z: 650 (MH⁺)

IR (ATR) cm⁻¹: 1676, 1512, 1425, 1203, 1176, 1124, 841, 800, 723, 619,517

Anal. (for C₃₀H₄₀N₁₀O₄S.0.50Et₂O.3.25H₂O) Calcd.: C, 51.56; H, 6.83; N,20.67. Found: C, 51.89; H, 6.48; N, 20.33.

Example 209(2-Amino-2-oxoethyl)[{[(1-{8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-2-yl}-4-piperidyl)oxy]carbonyl}amino]ethyl]dimethylammonium

(A)(2-Amino-2-oxoethyl)(2-{[({1-[3-[(E)-2-{1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido-[1,2-a]pyrimidin-2-yl]-4-piperidyl}oxy)carbonyl]amino}ethyl)dimethylammonium

1-(8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4-piperidyl-N-[2-(dimethylamino)ethyl]carbamate(97.0 mg, 0.128 mmol) and iodoacetamide (35.5 mg, 0.192 mmol) weredissolved in dimethylformamide (15 ml) and stirred overnight at roomtemperature. The reaction mixture was concentrated under reducedpressure, and the residue was purified by thin layer chromatography(silica gel, chloroform:methanol:water=8:3:0.5). The resulting solid waswashed with diethyl ether to obtain the title compound (78.1 mg, 75%) asyellow solid.

LRMS-ESI; m/z: 813 (MH⁺)

(B) (2-Amino-2-oxoethyl)[{[(1-{8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-4-piperidyl)oxy]carbonyl}amino]ethyl]dimethylammonium

(2-Amino-2-oxoethyl)(2-{[({1-[3-[(E)-2-{1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl]-4-piperidyl}oxy)carbonyl]amino}ethyl)dimethylammonium(78.1 mg, 0.0960 mmol) was dissolved in trifluoroacetic acid (20 ml),added with anisole (5.0 ml) and stirred at 60° C. for 5 hours. Thereaction mixture was cooled to room temperature and concentrated underreduced pressure, and the residue was purified by thin layerchromatography (silica gel, chloroform:methanol:water=8:3:0.5) to obtainthe title compound (78.1 mg) as orange solid.

m.p.: 213-217° C.

¹H-NMR (CD₃OD) δ: 1.34 (9H, s), 1.88 (2H, brs), 2.08 (2H, brs),3.30-3.34 (8H, m), 3.51 (2H, brs), 3.60 (2H, brs), 3.74 (2H, brs), 3.86(2H, brs), 4.87 (1H, brs), 6.56 (1H, s), 7.43 (1H, d, J=16.4Hz),7.87-7.94 (2H, m), 8.10 (1H, s), 8.92 (1H, d, J=7.10Hz)

LRMS-FAB; m/z: 693 (MH⁺)

HRMS-FAB (C₃₁H₄₁O₅N₁₂S); m/z: Calcd.: 693.3044 Found: 693.3020

IR (ATR) cm⁻¹: 1693, 1655, 1633, 1510, 1425, 1377, 1236, 1101, 1026

Example 210(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A)(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester

A solution of(E)-3-[8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (500 mg, 1.06 mmol) in a mixture of dimethylformamide(40 ml) and acetonitrile (20 ml) was added with diphenyl chlorophosphate(570 mg, 2.12 mmol) and diisopropylethylamine (725 μl, 4.24 mmol) withice cooling and stirred for 50 minutes. This solution was added with4-hydroxypiperidine (322 mg, 3.18 mmol) at 80-90° C., stirred for 90minutes, added with diisopropylethylamine (725 μl, 4.24 mmol) andfurther stirred for 2 hours. The reaction solution was cooled to roomtemperature and concentrated under reduced pressure, and the residue waspurified by column chromatography (silica gel, chloroform:ethylacetate=3:1-1:1) to obtain the title compound as yellow powder.

LRMS-ESI: 554 (MH⁺)

(B)(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (80.0 mg, 0.144 mmol) was dissolved in a solution ofhydrochloric acid in dioxane (4 N, 30 ml) and stirred at roomtemperature for 5 hours. The reaction mixture was concentrated underreduced pressure, and the residue was purified by thin layerchromatography (silica gel, lower layer of methylenechloride:methanol:water=7:3:1) to obtain the title compound (26.3 mg) asyellow powder.

m.p.: 215-218° C.

¹H-NMR (DMSO-d₆) δ: 1.30 (9H, s), 1.45-1.55 (2H, m), 1.86-1.89 (2H, m),3.32-3.50 (2H, m), 3.76-3.77 (1H, m), 3.84-3.68 (2H, m), 4.80 (1H, d,J=3.91Hz), 6.84 (1H, s), 6.91 (1H, d, J=15.4Hz), 7.43 (1H, d, J=15.6Hz),7.57-7.60 (1H, m), 8.16 (1H, d, J=1.22Hz), 8.88 (1H, d, J=7.32Hz)

LRMS-FAB; m/z: 498 (MH⁺)

HRMS-FAB (C₂₄H₂₈O₅S); m/z: Calcd.: 498.1811 Found: 498.1835

IR (ATR) cm⁻¹: 1666, 1520, 1441, 1365, 1292, 1217, 1196, 1103, 1068,739, 688, 476

Anal. (for C₂₄H₂₇N₅O₅S.0.75H₂O) Calcd.: C, 56.40; H, 5.62; N, 13.70.Found: C, 56.86; H, 5.48; N, 13.23.

Example 211(E)-3-[2-{4-[(Aminocarbonyl)oxy]piperidino}-8-({4-(t-butyl)-1,3-thiazol-2-yl}amino)carbonyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A)(E)-3-[2-{4-[(Aminocarbonyl)oxy]piperidino}-8-({4-(t-butyl)-1,3-thiazol-2-yl}amino)carbonyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester

(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (150 mg, 0.271 mmol) was suspended in ethyl acetate,added with trichloroacetyl isocyanate (161 μl, 1.36 mmol) and stirred atroom temperature for 2 hours and 30 minutes. The reaction mixture wasadded with a mixed solution of chloroform and methanol (10:1, 11 ml) toterminate the reaction and concentrated under reduced pressure to obtainorange amorphous solid. This solid was dissolved in a mixed solvent oftetrahydrofuran (40 ml) and water (20 ml), added with sodium formate(92.5 mg) and stirred at room temperature for 2 hours. The reactionmixture was concentrated under reduced pressure, and the residue waspurified by thin layer chromatography (silica gel,chloroform:methanol=9:1) to obtain the title compound (145 mg, 90%) asyellow amorphous solid.

LRMS-ESI; m/z: 597 (MH⁺)

(B) (E)3-[2-{4-[(Aminocarbonyl)oxy]piperidino}-8-({4-(t-butyl)-1,3-thiazol-2-yl}-amino)carbonyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(E)-3-[2-{4-[(Aminocarbonyl)oxy]piperidino}-8-({4-(t-butyl)-1,3-thiazol-2-yl}amino)carbonyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (145 mg, 0.243 mmol) was dissolved in a solution ofhydrochloric acid in dioxane (4 N, 50 ml) and stirred at roomtemperature for 4 hours. The reaction mixture was concentrated underreduced pressure, and the residue was purified by thin layerchromatography (silica gel, lower layer of methylenechloride:methanol:water=7:3:1) to obtain the title compound (60.6 mg) asyellow powder.

m.p.: 227-230° C.

¹H-NMR (DMSO-d₆) δ: 1.30 (9H, s), 1.65-1.67 (2H, m), 1.98 (2H, brs),3.34-3.43 (2H, m), 3.83-3.86 (2H, m), 4.77 (1H, m), 6.50 (2H, brs), 6.85(1H, s), 6.92 (1H, d, J=15.4Hz), 7.44 (1H, d, J=15.6Hz), 7.61 (1H, d,J=7.08Hz), 8.19 (1H, s), 8.90 (1H, d, J=7.81Hz)

LRMS-FAB; m/z: 541 (MH⁺)

IR (ATR) cm⁻¹: 1741, 1664, 1601, 1562, 1522, 1442, 1308, 1221, 1036,858, 742, 681, 633

Anal. (for C₂₅H₂₈N₆S.0.5H₂O) Calcd.: C, 54.63; H, 5.32; N, 15.29, S,5.8. Found: C, 54.71; H, 5.26; N, 15.14, S, 5.88.

Example 212(E)-3-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[4-({[(2-hydroxyethyl)amino]carbonyl}oxy)piperidino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A)(E)-3-[8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester

A solution of(E)-3-[8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (500 mg, 1.06 mmol) in a mixture of dimethylformamide(40 ml) and acetonitrile (20 ml) was added with diphenyl chlorophosphate(570 mg, 2.12 mmol) and diisopropylethylamine (548 mg, 4.24 mmol) withice cooling and stirred for 50 minutes. This solution was added with4-hydroxypiperidine (322 mg, 3.18 mmol) and diisopropylethylamine (548mg, 4.24 mmol) and stirred at 80-90° C. for 2 hours. The reactionsolution was concentrated under reduced pressure, and the resultingresidue was purified by column chromatography (silica gel,chloroform:ethyl acetate=3:1-1:1) to obtain the title compound (380 mg)as yellow powder.

LRMS-ESI; m/z: 554

(B)(E)-3-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[4-({[(2-hydroxyethyl)amino]carbonyl}oxy)piperidino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester

A solution of(E)-3-[8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (148 mg, 0.267 mmol) dissolved in a mixture ofmethylene chloride (30 ml) and tetrahydrofuran (10 ml) was added with asolution of N,N′-carbonyldiimidazole (261 mg, 1.60 mmol) in methylenechloride (20 ml) and stirred overnight at room temperature. The reactionmixture was washed with water and saturated brine and dried overanhydrous sodium sulfate, and the solvent was evaporated under reducedpressure.

The obtained yellow amorphous solid was dissolved in tetrahydrofuran(100 ml), added with triethylamine (81.1 mg, 0.801 mmol) and2-aminoethanol (32.6 mg, 0.534 mmol) and stirred overnight at roomtemperature. The reaction mixture was concentrated under reducedpressure, and the resulting residue was dissolved in chloroform,successively washed with 1 N aqueous hydrochloric acid, saturatedaqueous sodium hydrogencarbonate and saturated brine and dried overanhydrous sodium sulfate. The solvent was evaporated under reducedpressure, and the resulting residue was purified by thin layerchromatography (silica gel, methylene chloride:methanol=9:1) to obtainthe title compound (130 mg) as yellow amorphous solid.

LRMS-ESI; m/z: 641

(C)(E)-3-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[4-({[(2-hydroxyethyl)amino]carbonyl}oxy)piperidino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(E)-3-{8-({[4-(t-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[4-({[(2-hydroxyethyl)amino]carbonyl}oxy)piperidino]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester (130 mg, 0.203 mmol) was dissolved in a solution ofhydrochloric acid in dioxane (4 N, 30 ml) and stirred overnight at roomtemperature. The reaction mixture was concentrated under reducedpressure, and the resulting residue was purified by thin layerchromatography (silica gel, lower layer ofchloroform:methanol:water=7:3:1). The resulting solid was washed withmethanol to obtain the title compound (48.6 mg, 41%) as yellow powder.

m.p.: 231-234° C.

¹H-NMR (DMSO-d₆) δ: 1.30 (9H, s), 1.65-1.67 (2H, m), 1.97 (2H, brs),3.04 (2H, dd, J=6.10, 12.2Hz), 3.32-3.44 (6H, m), 3.81 (2H, brs), 4.61(1H, t, J=5.49Hz), 4.81 (1H, brs), 6.86 (1H, s), 6.92 (1H, d, J=15.6Hz),7.06 (1H, t, J=5.86Hz), 7.44 (1H, d, J=15.6Hz), 7.61 (1H, dd, J=1.83,7.45Hz), 8.19 (1H, s), 8.90 (1H, d, J=7.33Hz)

LRMS-ESI; m/z: 585 (MH⁺)

IR (ATR) cm⁻¹: 2962, 1662, 1520, 1427, 1311, 1221, 1138, 1066, 1014,860, 733, 471

Anal. (for C₂₇H₃₂N₆O₇S.0.75H₂O) Calcd.: C, 54.22; H, 5.64; N, 14.05; S,5.36. Found: C, 54.35; H, 5.42; N, 13.99; S, 5.34.

Example 213(E)-3-[2-{(3R)-3-[(Aminosulfonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A)(E)-3-[2-{(3R)-3-[(Aminosulfonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester

A solution of(E)-3-{8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid t-butyl ester (178 mg, 0.321 mmol), triethylamine (260 mg, 2.57mmol) and 4-dimethylaminopyridine (19.7 mg, 0.161 mmol) in methylenechloride (50 ml) was added with a solution of sulfamoyl chloride inmethylene chloride (1.18 mmol/ml, 1.08 ml) with ice cooling and stirredat room temperature for 24 hours. The reaction solution was washed withwater, and the aqueous layer was extracted with chloroform three times.The combined organic layer was concentrated under reduced pressure. Theresulting residue was purified by thin layer chromatography(n-hexane:ethyl acetate=1:3) to obtain the title compound (161 mg, 79%)as yellow solid.

¹H-NMR (CDCl₃) δ: 1.36 (9H, s), 1.45 (9H, s), 1.63 (2H, brs), 2.00 (2H,brs), 3.49 (2H, brs), 2.72-3.75 (2H, m), 4.81 (1H, brs), 6.54 (1H, s),7.00-7.04 (1H, m), 7.36 (1H, brs), 7.54 (1H, d, J=7.57Hz), 7.90 (1H, d,J=1.22Hz), 9.02 (1H, d, J=7.33Hz)

LRMS-ESI; m/z: 633 (MH⁺)

(B)(E)-3-[2-{(3R)-3-[(Aminosulfonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(E)-3-[2-{(3R)-3-[(Aminosulfonyl)oxy]hexahydro-1-pyridinyl}-8-({[4-(t-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid t-butyl ester (733 mg, 1.16 mmol) was dissolved in a solution ofhydrochloric acid indioxane (4 N, 100 ml) and stirred overnight at roomtemperature. The reaction mixture was concentrated under reducedpressure, and the residue was purified by column chromatography (silicagel, methylene chloride:methanol=9:1,chloroform:methanol:water=8:3:0.5). The obtained yellow solid was washedwith a mixed solvent of diethyl ether, n-hexane and methanol to obtainthe title compound (369 mg, 55%) as yellow powder.

m.p.: 222° C. (decomp.)

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.76-1.77 (1H, m), 1.95-2.03 (2H, m),2.13-2.17 (1H, m), 3.61 (2H, t, J=5.27Hz), 3.65-3.70 (1H, m), 4.04-4.08(1H, m), 4.71-4.79 (1H, m), 6.74 (1H, s), 7.07 (1H, d, J=15.7Hz),7.59-7.63 (2H, m), 8.02-8.08 (1H, m), 8.95-8.97 (1H, m)

IR (ATR) cm⁻¹: 3332, 3103, 2960, 1668, 1512, 1441, 1365, 1271, 1173,1099, 930, 901, 864, 829, 741, 687, 552

LRMS-FAB; m/z: 577 (MH⁺)

Anal. (for C₂₄H₂₈N₆O₇S₂-1.75H₂O) Calcd.: C, 47.40; H, 5.22; N, 13.82.Found: C, 47.21; H, 5.04; N, 13.47.

Example 231(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3,4-dihydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-3-formyl-2-hydroxy-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxyamide

Dimethylformamide (5 ml) was added dropwise with phosphorus oxychloride(54 μl, 0.58 mmol, 2 eq) with ice cooling and stirred at roomtemperature for 20 minutes. This mixture was added withN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2,4-dioxo-3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxamide(100 mg, 0.29 mmol) at 0° C., stirred at room temperature for 30 minutesand then stirred at 80° C. for 20 minutes. After completion of thereaction, the reaction solution returned to room temperature was addedwith water and saturated aqueous sodium hydrogencarbonate (pH 5-6) andextracted with chloroform. The collected organic layer was dried overmagnesium sulfate, and the solvent was concentrated under reducedpressure. The obtained oily title compound was used in the followingreaction without being purified.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.31 (9H, s), 6.63 (1H, s), 8.03 (2H, m), 9.16(1H, brd), 10.16 (1H, brd).

EI-MS; m/s: 373 (M⁺+1).

(B) tert-Butyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-3-formyl-2-hydroxy-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxamide(108 mg, 0.29 mmol) was dissolved in tetrahydrofuran (5 ml), added with(tert-butoxycarbonylmethylene)triphenylphosphorane (165 mg, 0.44 mmol)at room temperature and stirred at the same temperature for 2 hours.After completion of the reaction, the solvent was concentrated underreduced pressure, and the obtained oily substance was purified by thinlayer silica gel column chromatography (lower layer ofchloroform:methanol:water=8:6:1) to obtain the title compound (63 mg,46% for the two steps) as a yellow substance.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.38 (9H, s), 1.53 (9H, s), 6.63 (1H, s), 7.05(1H, d, J=15.87Hz), 8.00 (1H, d, J=15.87Hz), 8.01 (1H, d, J=7.08Hz),8.06 (1H, s), 9.18 (1H, d, J=7.08Hz).

EI-MS; m/s: 471 (M⁺+1).

(C) tert-Butyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3,4-dihydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

tert-Butyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(63 mg, 0.13 mmol) was added with 1 ml each of dimethylformamide andacetonitrile, cooled to −10° C. with ice and then added dropwise withdiphenylphosphonyl chloride (56 μl, 0.26 mmol, 2 eq) anddiisopropylethylamine (93 μl, 0.52 mmol, 4 eq). The mixture was stirredat the same temperature for 10 minutes, added with3,4-dihydroxypiperidine hydrochloride (31 mg, 0.20 mmol, 1.5 eq) anddiisopropylethylamine (50 μl, 0.26 mmol, 2 eq), stirred for 15 minutesand further stirred at room temperature for 20 minutes. Then the mixturewas heated to 90° C., stirred for 1 hour and 30 minutes, added with3,4-dihydroxypiperidine hydrochloride (20 mg) and diisopropylethylamine(25 μl) and further stirred for 40 minutes. After completion of thereaction, the reaction solution was added with water and extracted withchloroform. The collected organic layer was dried over magnesiumsulfate, and then the solvent was concentrated under reduced pressure.The obtained oily substance was purified by thin layer silica gel columnchromatography (chloroform:methanol=30:1) to obtain the yellow titlecompound (35 mg, 45%).

¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 1.52 (9H, s), 1.69 (1H, m), 1.88 (1H,m), 3.22 (1H, m), 3.45 (3H, m), 3.82 (1H, m), 3.90 (1H, m), 6.58 (1H,s), 7.06 (1H, d, J=15.87Hz), 7.43 (1H, d, J=15.87Hz), 7.53 (1H, dd,J=7.32, 1.47Hz), 8.03 (1H, s), 8.95 (1H, d, J=7.32Hz).

EI-MS; m/s: 570 (M⁺+1).

(D)(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3,4-dihydroypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

tert-Butyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3,4-dihydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl-2-propenoate(35 mg, 0.06 mmol) was added with 4 N solution of hydrochloric acid indioxane (3 ml) at room temperature and stirred at the same temperaturefor 4 hours. After completion of the reaction, the solvent wasconcentrated under reduced pressure, and the obtained oily substance waspurified by thin layer silica gel column chromatography(chloroform:methanol=10:1, developed 4 times) to obtain the titlecompound (18 mg, 26% for the two steps) as yellow crystals.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.36 (9H, s), 1.87 (1H, m), 1.99 (1H, m), 3.60(1H, m), 3.77 (3H, m), 3.88 (1H, m), 3.96 (1H, m), 6.64 (1H, s), 7.05(1H, d, J=15.68Hz), 7.56 (1H, d, J=7.35Hz), 7.62 (1H, d, J=15.68Hz),8.06 (1H, s), 8.95 (1H, d, J=7.35Hz).

m.p.: 182-189° C.

ES-MS; m/s: 514 (M⁺+1).

Example 232(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-3-formyl-2-hydroxy-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxyamide

Dimethylamide (15 ml) was added dropwise with phosphorus oxychloride (97μl, 1.04 mmol, 1.2 eq) with ice cooling and stirred at room temperaturefor 30 minutes. The reaction solution was cooled with ice again, thenadded withN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2,4-dioxo3,4-dihydro-2H-pyrido[1,2-a]pyrimidine-8-carboxamide(300 mg, 0.87 mmol) at 80-90° C. and stirred for 45 minutes. Thereaction solution was returned to room temperature, then neutralized byadding saturated aqueous sodium hydrogencarbonate and extracted withchloroform. The resulting organic layer was dried over magnesiumsulfate, and the solvent was further concentrated under reduced pressureto obtain a crude product (360 mg) as a yellow oily substance.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.31 (9H, 8), 6.63 (1H, a), 8.03 (2H, m), 9.16(1H, brd), 10.16 (1H, brd).

EI-MS; m/s: 373 (M⁺+1).

(B) tert-Butyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino)carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-3-formyl-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(324 mg, 0.87 mmol) was dissolved in tetrahydrofuran (20 ml) anddimethylformamide (2 ml), added with(tert-butoxycarbonylmethylene)triphenylphosphorane (490 mg, 1.31 mmol,1.5 eq) and stirred at room temperature for 3 hours. Then the mixturewas further added with 250 mg of(tert-butoxycarbonylmethylene)triphenylphosphorane, and 1 hour afterthat, further added with 300 mg of(tert-butoxycarbonylmethylene)triphenylphosphorane. After the reactionmixture was stirred for 10 hours, the solvent was concentrated underreduced pressure, and the obtained oily substance was purified by silicagel column chromatography (chloroform:methanol=100:1→20:1) to obtain thetitle compound (368 mg) as a mixture with triphenylphosphine oxide.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.38 (9H, a), 1.53 (9H, a), 6.63 (1H, a), 7.05(1H, d, J=15.87Hz), 8.00 (1H, d, J=15.87Hz), 8.01 (1H, d, J=7.08Hz),8.06 (1H, 8), 9.18 (1H, d, J=7.08Hz).

EI-MS; m/s: 471 (M⁺+1).

(C) tert-Butyl(E)3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3,4-dihydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

tert-Butyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(367 mg, 0.78 mmol) was dissolved in dimethylformamide and acetonitrile(8 ml each), cooled to −10° C. with ice and added dropwise withphosphonyl chloride (0.3 ml, 1.56 mmol, 2 eq) and diisopropylamine (0.5ml, 3.12 mmol, 4 eq). The reaction mixture was stirred at the sametemperature for 10 minutes, added with 3,4-dihydroxypiperidinehydrochloride (180 mg) and diisopropylamine (0.25 ml), stirred at roomtemperature for 5 minutes and then stirred at 90° C. for 1 hour. Thenthe reaction mixture was added with 3,4-dihydroxypiperidinehydrochloride (80 mg) and diisopropylamine (0.25 ml) and further stirredat 90° C. for 1 hour. The reaction solution was returned to roomtemperature, added with water, extracted with chloroform, and theorganic solvent was dried over magnesium sulfate. The solvent wasconcentrated under reduced pressure, and the resulting crude product waspurified by silica gel column chromatography(chloroform:methanol=100:1→20:1) to obtain the title compound (190 mg)with contained impurities.

¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 1.52 (9H, s), 1.69 (1H, m), 1.88 (1H,m), 3.45 (2H, m), 3.82 (1H, m), 3.90 (1H, m), 4.11 (1H, s), 4.18 (1H, d,J=13.92Hz), 6.58 (1H, s), 7.06 (1H, d, J=15.87Hz), 7.43 (1H, d,J=15.87Hz), 7.53 (1H, dd, J=7.32, 1.47Hz), 8.03 (1H, s), 8.95 (1H, d,J=7.32Hz).

EI-MS; m/s: 570 (M⁺+1).

(D) tert-Butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

tert-Butyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3,4-dihydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate (213 mg, 0.37 mmol) dissolvedin ethyl acetate (5 ml) was added with trichloroacetyl isocyanate (230μl, 1.85 mmol, 5 eq) with ice cooling and stirred at the sametemperature for 30 minutes. Then the mixture was further added with 0.5ml of the isocyanate and after 30 minutes, further added with 0.5 ml ofthe same. After the reaction mixture was stirred for 30 minutes, thesolvent was concentrated under reduced pressure, and the obtained oilysubstance was added with methanol (5 ml), water (1 ml) and sodiumformate (330 mg) and stirred at room temperature. After 40 minutes, thereaction mixture was further added with sodium formate (500 mg), andafter 20 minutes, further added with sodium formate (500 mg). After 30minutes, the reaction mixture was further added with sodium formate (500mg), water (1 ml) and methanol (0.5 ml) and stirred for 30 minutes. Thenthe reaction solution was added with water and extracted withchloroform. The collected organic layer was dried over magnesiumsulfate, and the solvent was concentrated under reduced pressure. Theobtained oily substance was purified by silica gel column chromatographyto obtain the title compound (77 mg).

¹H-NMR (CDCl₃) δ: 1.31 (9H, s), 1.48 (9H, s), 1.94 (1H, m), 2.16 (1H,m), 3.40 (1H, m), 3.61 (1H, m), 3.83 (1H, m), 4.01 (1H, m), 5.09 (2H,s), 6.62 (1H, s), 7.11 (1H, d, J=15.87Hz), 7.57 (1H, m), 7.58 (1H, d,J=15.87Hz), 8.21 (1H, s), 9.00 (1H, d, J=7.32Hz).

EI-MS; m/s: 656 (M⁺+1).

(E)(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

tert-Butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate(76.9 mg, 0.1173 mmol) was added with 4 N solution of hydrochloric acidin dioxane (6 ml) at room temperature and stirred at the sametemperature for 3 hours. After completion of the reaction, the solventwas concentrated under reduced pressure, and the obtained oily substancewas purified by thin layer silica gel column chromatography (lower layerof chloroform:methanol:water=8:3:1) to obtain the title compound (30 mg,6% for the six steps) as yellow crystals.

¹H-NMR (CD₃OD) δ: 1.36 (9H, s), 1.97 (1H, m), 2.12 (1H, m), 3.55 (1H,m), 3.71 (1H, d, J=13.92Hz), 3.89 (1H, m), 4.21 (1H, m), 5.05 (2H, s),6.65 (1H, s), 7.06 (1H, d, J=15.63Hz), 7.61 (1H, m), 7.62 (1H, d,J=15.63Hz), 8.10 (1H, s), 8.96 (1H, d, J=7.32Hz).

m.p.: 193-199° C. (decomp.)

FAB-MS; m/s: 600 (M⁺+1).

Example 233(3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinylcarbamate

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido-[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(100 mg, 0.18 mmol) was dissolved in dimethylformamide (2 ml) andacetonitrile (2 ml) and cooled to −10° C. with ice. The reaction mixturewas added dropwise with diphenylphosphonyl chloride (74 μl, 0.36 mmol, 2eq) and diisopropylamine (125 μl, 0.72 mmol, 4 eq) and stirred at thesame temperature for 20 minutes. Then the reaction mixture was addedwith (R)-(+)-3-hydroxypiperidine hydrochloride (40 mg, 0.2685 mmol, 1.5eq) and diisopropylamine (63 μl, 0.36 mmol, 2 eq) at the sametemperature, warmed to room temperature and then to 80° C., and after 1hour, further added with piperidine (45 mg) and diisopropylamine (70μl). Further, after stirred for 1 hour, the reaction solution wasreturned to room temperature, added with water and extracted withchloroform, and the organic layer was dried over magnesium sulfate. Thesolvent was concentrated under reduced pressure, and the obtained oilysubstance was purified by silica gel column chromatography(chloroform→chloroform:methanol=100:1→80:1→50:1) to obtain the titlecompound (143 mg, almost quantitative) as a yellow oily substance withcontained dimethylformamide. ¹H-NMR (CDCl₃) δ: 1.37 (9H, s), 1.48 (2H,m), 1.84 (2H, m), 3.32 (1H, m), 3.54 (2H, m), 3.64 (1H, m), 3.78 (3H,s), 4.07 (1H, s), 5.51 (2H, s), 6.54 (1H, s), 6.88 (2H, d, J=8.82Hz),7.34 (2H, d, J=8.82Hz), 7.47 (1H, dd, J=7.59, 1.71Hz), 8.00 (1H, s),8.03 (2H, s), 8.91 (1H, d, J=7.59Hz).

ES-MS; m/s: 642 (M⁺+1)

(B)(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)hexahydro-3-pyridinylcarbamate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(143 mg, 0.22 mmol) was dissolved in ethyl acetate (3 ml), cooled to 0°C. with ice, added with trichloro-isocyanate (45 μl, 0.36 mmol, 1.6 eq),stirred at the same temperature for 20 minutes, further added withtrichloro-isocyanate (45 μl) and stirred for 15 minutes. Aftercompletion of the reaction, the solvent was concentrated under reducedpressure, and the obtained oily substance was added with methanol (4ml), water (0.6 ml) and sodium formate (145 mg) at room temperature andstirred at the same temperature for 30 minutes. Then the reactionmixture was added with sodium formate (170 mg), after 30 minutes,further added with sodium formate (170 mg), water (0.5 ml) and methanol(1 ml) and stirred for 10 hours. The reaction mixture was added withwater and extracted with chloroform, and the organic layer was driedover magnesium sulfate. The solvent was concentrated under reducedpressure, and the obtained oily substance was purified by thin layersilica gel column chromatography (chloroform:methanol=15:1) to obtainthe yellow title compound (144 mg) with contained impurities.

¹H-NMR (CDCl₃) δ: 1.32 (9H, s), 1.49 (1H, m), 1.84 (3H, m), 3.42 (2H,m), 3.78 (3H, s), 3.86 (1H, d, J=14.40Hz), 3.96 (1H, d, J=10.99Hz), 4.90(1H, s), 5.53 (2H, s), 6.62 (1H, s), 6.89 (2H, d, J=8.79Hz), 7.34 (2H,d, J=8.79Hz), 7.59 (1H, d, J=7.32Hz), 7.98 (2H, s), 8.12 (1H, s), 8.99(1H, d, J=7.32Hz).

EI-MS; m/s: 685 (M⁺+1)

(C)(3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinylcarbamate

(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylcarbamate(144 mg, 0.21 mmol) was added with trifluoroacetic acid (10 ml) andstirred at room temperature for 17 hours. The solvent was concentratedunder reduced pressure, and the obtained oily substance was purified bythin layer silica gel column chromatography (lower layer ofchloroform:methanol:water=8:3:1) to obtain the title compound (39 mg,39% for the three steps) as yellow crystals.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.36 (9H, s), 1.71 (1H, m), 1.97 (3H, m), 3.60(1H, m), 3.76 (2H, m), 3.89 (1H, m), 4.87 (1H, s), 6.64 (1H, s), 7.62(1H, d, J=7.31Hz), 7.68 (1H, d, J=15.60Hz), 7.86 (1H, d, J=15.60Hz),8.12 (1H, s), 8.98 (1H, d, J=7.31Hz).

ES-MS; m/s: 565 (M⁺+1)

m.p.: 206-217.8° C.

FAB-MS; m/s: 565 (MH⁺)

Example 234N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidine-8-carboxyamide

(A) tert-Butyl 3,4-di[(aminocarbonyl)oxy]-1-piperidincarboxylate

tert-Butyl 3,4-dihydroxy-1-piperidinecarboxylate (200 mg, 0.92 mmol) wasdissolved in ethyl acetate (10 ml) and cooled to 0° C. with ice. Thesolution was added dropwise with trichloro-isocyanate (0.2 ml, 1.94mmol, 2 eq) and stirred at the same temperature for 30 minutes. Then thereaction mixture was added with trichloro-isocyanate (0.2 ml), and after40 minutes, further added with 0.25 ml of trichloro-isocyanate. Afterstirred for 30 minutes, the reaction solvent was concentrated underreduced pressure, and the obtained oily substance was added withmethanol (9 ml), water (1.8 ml) and sodium formate (750 mg) at roomtemperature and stirred at the same temperature for 1 hour. Then thereaction mixture was added with sodium formate (750 mg) and furtherstirred for 1 hour. After completion of the reaction, the reactionsolution was added with water and extracted with chloroform, and theorganic layer was dried over magnesium sulfate. The solvent wasevaporated under reduced pressure, and the obtained oily substance waspurified by silica gel column chromatography(chloroform→chloroform:methanol=80:1→50:1→20:1) to obtain the titlecompound (277.7 mg, 100%).

¹H-NMR (CD₃OD) δ: 1.46 (9H, s), 1.78 (1H, m), 1.93 (1H, m), 3.09 (1H,m), 3.28 (1H, m), 3.89 (1H, m), 4.07 (1H, m), 4.85 (2H, m).

EI-MS; m/s: 304 (M⁺+1)

(B) 3,4-Di[(aminocarbonyl)oxy]piperidine

tert-Butyl 3,4-di[(aminocarbonyl)oxy]-1-piperidinecarboxylate (278 mg,0.92 mmol) was added with 4 N solution of hydrochloric acid in dioxane(25 ml) at room temperature and stirred at the same temperature for 11hours. The reaction solution was concentrated under reduced pressure toobtain the title compound (248 mg, 100%) as white crystals.

¹H-NMR (CD₃OD) δ: 2.07 (1H, m), 2.21 (1H, m), 3.17 (1H, m), 3.36 (3H,m), 4.98 (1H, m), 5.18 (1H, m).

EI-MS; m/s: 204 (M⁺+1)

(C)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-3-(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(150 mg, 0.27 mmol) was dissolved in acetonitrile (3 ml) anddimethylformamide (3 ml) and cooled to −10° C. with ice. The solutionwas added with diphenylphosphonyl chloride (111 μl, 0.54 mmol, 2 eq) andthen added with diisopropylamine (185 μl, 1.08 mmol, 4 eq). The mixturewas stirred at the same temperature for 10 minutes, then further addedwith diphenylphosphonyl chloride (60 μl) and diisopropylamine (90 μl)and further stirred for 10 minutes. Then the mixture was added with3,4-di[(aminocarbonyl)oxy]piperidine hydrochloride (95 mg, 0.41 mmol,1.5 eq) and diisopropylamine (95 μl, 0.54 mmol, 2 eq), heated to 80° C.and stirred for 40 minutes. The mixture was added with3,4-di[(aminocarbonyl)oxy]piperidine hydrochloride (60 mg) anddiisopropylamine (40 μl) and after 30 minutes, further added with3,4-di[(aminocarbonyl)oxy]piperidine hydrochloride (70 mg) anddiisopropylamine (80 μl). After stirred for 1 hour, the reactionsolution was added with water and extracted with chloroform, and theorganic layer was dried over magnesium sulfate. The solvent wasconcentrated under reduced pressure, and the obtained oily compound waspurified by thin layer silica gel column chromatography(chloroform:methanol=10:1) to obtain the title compound (67 mg, 34%) asyellow crystals.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.36 (9H, s), 1.93 (1H, m), 2.10 (1H, m), 3.52(1H, m), 3.65 (1H, m), 3.80 (3H, s), 3.97 (1H, m), 4.19 (1H, m), 5.05(2H, m), 5.56 (2H, s), 6.62 (1H, s), 6.91 (2H, d, J=8.78Hz), 7.36 (2H,d, J=8.78Hz), 7.62 (1H, d, J=7.56Hz), 7.73 (1H, d, J=15.36Hz), 7.90 (1H,d, J=15.36Hz), 8.11 (1H, s), 9.01 (1H, d, J=7.56Hz).

EI-MS; m/s: 744 (M⁺+1)

(D)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-3-(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(67 mg, 0.09 mmol) was added with trifluoroacetic acid (10 ml) at roomtemperature and stirred at the same temperature for 15 hours. Then thesolvent of the reaction solution was concentrated under reducedpressure, and the obtained oily substance was purified by thin layersilica gel column chromatography (lower layer ofchloroform:methanol:water=8:3:1) to obtain the title compound (30 mg,53%) as orange crystals.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.95 (1H, m), 2.15 (1H, m), 3.57 (1H,m), 3.72 (1H, m), 4.02 (1H, m), 4.25 (1H, m), 5.02 (2H, m), 6.75 (1H,s), 7.52 (2H, d, J=16.36Hz), 7.59 (1H, d, J=7.32Hz), 7.89 (1H, d,J=16.36Hz), 7.08 (1H, s), 8.98 (1H, d, J=7.32Hz).

m.p.: 184-214° C. (decomp.)

EI-MS; m/s: 624 (M⁺+1)

FAB-MS; m/s: 624 (MH⁺)

Example 235(E)-3-[8-{[(4-Cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A) tert-Butyl(E)-3-{8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl(E)-3-(8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin3-yl)-2-propenoate(50 mg, 0.11 mmol) was dissolved in acetonitrile (1 ml) anddimethylformamide (1 ml), cooled to −10° C. with ice, added withdiphenylphosphonyl chloride (46 μl, 0.22 mmol, 2 eq) anddiisopropylamine (80 μl, 0.44 mmol, 4 eq) and stirred at the sametemperature for 10 minutes. Then the reaction mixture was added withdiphenylphosphonyl chloride (40 μl) and diisopropylamine (60 μl) andfurther stirred for 10 minutes. The reaction mixture was added with3,4-di[(aminocarbonyl)oxy]piperidine hydrochloride (40 mg, 0.17 mmol,1.5 eq) and diisopropylamine (40 μl) and heated to 90° C. After stirredfor 30 minutes, the mixture was added with3,4-di[(aminocarbonyl)oxy]piperidine hydrochloride (100 mg) anddiisopropylamine (100 μl) and further stirred for 30 minutes. Thereaction solution was returned to room temperature, added with water andextracted with chloroform, and the organic layer was dried overmagnesium sulfate. The solvent was concentrated under reduced pressure,and the obtained oily compound was purified by thin layer silica gelcolumn chromatography (chloroform:methanol=10:1) to obtain the yellowtitle compound (23 mg, 32%).

¹H-NMR (CD₃OD/CDCl₃) δ: 1.52 (9H, s), 1.95-2.39 (8H, m), 3.45-3.70 (4H,m), 3.86 (1H, m), 5.03 (1H, m), 5.09 (1H, m), 6.63 (1H, s), 7.08 (1H, d,J=15.63Hz), 7.51 (1H, d, J=15.63Hz), 7.56 (1H, d, J=7.56Hz), 8.08 (1H,s), 8.98 (1H, d, J=7.56Hz).

EI-MS; m/s: 654 (M⁺+1)

(B)(E)-3-[8-{[(4—Cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

tert-Butyl(E)-3-{8-{[(4-cyclobutyl-1,3-thiazol-2-yl)amino]carbonyl}-2-{3,4-di[(aminocarbonyl)oxy]piperidino}-4-oxo-4H-pyrido[1,2-a]pyrimidin-.3-yl}-2-propenoate(23 mg, 0.04 mmol) was added with 4 N solution of hydrochloric acid indioxane (3 ml) and stirred at room temperature for 3 hours. The solventwas concentrated under reduced pressure, and the obtained oily compoundwas purified by thin layer silica gel column chromatography (lower layerof chloroform:methanol:water=8:3:1) to obtain the title compound (11 mg,52%) as yellow crystals.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.94-2.37 (8H, m), 3.40-3.60 (4H, m), 3.86 (1H,m), 5.08 (2H, m), 6.63 (1H, s), 7.10 (1H, m), 7.63 (2H, m), 8.12 (1H,s), 8.98 (1H, m).

m.p.: 202-210° C. (decomp.)

EI-MS; m/s: 598 (M⁺+1)

FAB-MS; m/s: 598 (MH⁺)

Example 236[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-carboxy-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)-oxy]carbonyl}amino)ethyl](trimethyl)ammonium

(A) tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(2-chloroethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(237 mg, 0.43 mmol) was added with dichloromethane (4 ml) and pyridine(50 μl), added with 2-chloroethyl isocyanate (73 μl, 0.86 mmol) for 40minutes, stirred at room temperature and refluxed by heating at 60° C.for 1 hour. The mixture was added with 2-chloroethyl isocyanate (73 μl,0.86 mmol) and further refluxed by heating for 24 hours. Then thereaction mixture was returned to room temperature and added with waterto terminate the reaction. The reaction solution was extracted withchloroform, and the collected organic layer was dried over magnesiumsulfate. The solvent was concentrated under reduced pressure, and theresulting crude product was purified by thin layer silica gelchromatography (chloroform:methanol=3:1) to obtain the target compound(287 mg).

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.52 (9H, s), 1.74-1.92 (6H, m),3.20-3.69 (6H, m), 4.87 (1H, 9), 6.61 (1H, a), 7.15 (1H, d, J=14.67Hz),7.48 (1H, d, J=7.34Hz), 7.55 (1H, d, J=14.67Hz), 7.95 (1H, s), 8.99 (1H,d, J=7.34Hz).

EI-MS; m/s: 660 (M⁺+1)

(B) tert-Butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[({[2-(dimethylamino)ethyl]amino)carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino)carbonyl)-2-[(3R)-3-({[(2-chloroethyl)amino]carbonyl]oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(196 mg, 0.30 mmol) was added with dimethylformamide (2 ml), then addedwith a dimethylamine solution in tetrahydrofuran (2.0 M, 0.75 ml, 1.50mmol, 5 eq) and heated to 80° C. Then a dimethylamine solution intetrahydrofuran was occasionally added until completion of the reaction,and 9.25 ml was used in total. The reaction was performed for 30 hours.The reaction solution was added with saturated brine and extracted withchloroform, and the resulting organic layer was dried over magnesiumsulfate. The solvent was concentrated under reduced pressure, and theresulting crude product was purified by thin layer silica gelchromatography (chloroform:methanol=20:1) to obtain the title compound(55 mg).

¹H-NMR (CDCl₃) δ: 1.34 (9H, 8), 1.52 (9H, a), 1.65 (1H, m), 1.94 (3H,m), 2.22 (6H, 8), 2.34 (1H, m), 2.47 (1H, m), 3.20 (1H, m), 3.31 (1H,m), 3.49 (1H, m), 3.70 (2H, m), 3.85 (1H, d, J=12.72Hz), 4.96 (1H, s),6.61 (1H, s), 7.10 (1H, d, J=15.41Hz), 7.45 (1H, d, J=7.34Hz), 7.55 (1H,d, J=15.41Hz), 7.94 (1H, s), 8.98 (1H, d, J=7.34Hz).

EI-MS; m/s: 668 (M⁺+1).

(C)(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[({[2-(dimethylamino)ethyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

tert-Butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[({[2-(dimethylamino)ethyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate(55 mg, 0.08 mmol) was added with 4 N hydrochloric acid in dioxane (3ml) at room temperature and stirred at the same temperature for 1 hour30 minutes. After completion of the reaction, the reaction mixture wasconcentrated under reduced pressure, and the obtained oily crude productwas purified by thin layer silica gel chromatography (lower layer ofchloroform:methanol:water=8:3:1) to obtain the title compound (21 mg, 8%for the three steps).

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.71 (2H, m), 1.95 (1H, d, J=12.74Hz),2.11 (1H, d, J=12.74Hz), 2.57 (6H, s), 2.66 (1H, m), 2.88 (1H, m), 2.98(1H, t, J=11.27Hz), 3.10 (1H, d, J=13.72Hz), 3.21 (1H, m), 3.70 (1H, m),3.84 (1H, d, J=13.72Hz), 4.13 (1H, d, J=11.27Hz), 4.98 (1H, s), 6.19(1H, d, J=6.12Hz), 6.62 (1H, s), 6.95 (1H, d, J=15.43Hz), 7.46 (1H, dd,J=7.35, 1.96Hz), 7.80 (1H, d, J=15.43Hz), 7.87 (1H, s), 9.03 (1H, d,J=7.35Hz).

EI-MS; m/s: 612 (M⁺+1).

(D)[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-carboxy-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](trimethyl)ammonium

(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[({[2-(dimethylamino)ethyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid (21 mg, 0.03 mmol) was added with N,N-dimethylformamide (4 ml) andmethyl iodide (43 μl, 0.68 mmol, 20 eq) and left stand in therefrigerator for 20 hours. Then the mixture was further added withmethyl iodide (50 μl, 0.79 mmol, 23 eq) and left stand in therefrigerator for 24 hours. After completion of the reaction wasconfirmed, the solvent was concentrated under reduced pressure, and theresulting crude product was purified by liquid chromatography(acetonitrile:water=30:70, 0.1% formic acid solution, flow rate 10cc/min) to obtain the title compound (17 mg, 79%). (

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.67 (1H, brd), 2.01 (3H, brd), 3.19(9H, s), 3.41-3.71 (6H, m), 3.92 (1H, m), 4.04 (1H, m), 4.85 (1H, brd),6.75 (1H, s), 7.08 (1H, brd), 7.62 (2H, m), 8.00 (1H, s), 8.95 (1H, d,J=7.34Hz).

ES-MS; m/s: 626 (M⁺).

Example 237[3-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-carboxy-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl](trimethyl)ammonium

(A) tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(3-chloropropyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate

tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(682 mg, 1.23 mmol) was added with tetrahydrofuran (20 ml) and pyridine(155 μl), then added with 3-chloropropyl isocyanate (265 μl, 2.46 mmol)and refluxed by heating at 80° C. for 3 hours. Then 3-chloropropylisocyanate was added until completion of the reaction, and 2.1 ml (17.2mmol, 14 eq) was used in total. The reaction mixture was stirred for 3days. The reaction mixture was returned to room temperature and addedwith saturated aqueous sodium hydrogencarbonate to terminate thereaction. The reaction mixture was extracted with chloroform, and thecollected organic layer was dried over magnesium sulfate. The solventwas concentrated under reduced pressure, and the resulting crude productwas purified by silica gel chromatography(chloroform→chloroform:methanol=100:1) to obtain the target compound(522.5 mg, 63%).

¹H-NMR (CDCl₃) δ: 1.33 (9H, s), 1.52 (9H, s), 1.69-2.04 (6H, m),3.19-3.94 (8H, m), 4.86 (1H, s), 5.80 (1H, brd), 6.59 (1H, s), 7.11 (1H,d, J=15.36Hz), 7.50 (1H, d, J=7.31Hz), 7.68 (1H, d, J=15.36Hz), 7.97(1H, s), 8.98 (1H, d, J=7.31Hz).

ES-MS; m/s: 675 (M⁺+1).

(B) tert-Butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[({[3-(dimethylamino)propyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate

tert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(3-chloropropyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(258 mg, 0.4 mmol) was added with dimethylformamide (4 ml), then addedwith a dimethylamine solution in tetrahydrofuran (2.0 M, 0.75 ml, 1.50mmol, 5 eq) and heated to 80° C. Then the dimethylamine solution intetrahydrofuran was occasionally added until completion of the reaction,and 17 ml (34 mmol, 85 eq) was used in total. The reaction was performedfor 48 hours. The reaction solution was added with saturated brine andextracted with chloroform. The collected organic layer was dried overmagnesium sulfate. The solvent was concentrated under reduced pressure,and the resulting crude product was purified by thin layer silica gelchromatography (chloroform:methanol=10:1) to obtain the title compound(46 mg, 18%).

¹H-NMR (CDCl₃) δ: 1.33 (9H, s), 1.52 (9H, s), 1.72 (3H, brd), 1.92 (3H,brd), 2.26 (6H, s), 2.41 (2H, m), 3.26 (2H, m), 3.49 (1H, m), 3.73 (2H,m), 3.85 (1H, m), 4.90 (1H, s), 5.84 (1H, brd), 6.60 (1H, s), 7.11 (1H,d, J=15.36Hz), 7.47 (1H, d, J=7.56Hz), 7.57 (1H, d, J=15.36Hz), 7.96(1H, s), 8.98 (1H, d, J=7.56Hz).

EI-MS; m/s: 682 (M⁺+1).

(C)(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[({[3-(dimethylamino)propyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

tert-Butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[({[3-(dimethylamino)propyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate(46 mg, 0.07 mmol) was added with 4 N hydrochloric acid in dioxane (5ml) at room temperature and stirred for at the same temperature 1 hourand 30 minutes. After completion of the reaction, the reaction mixturewas concentrated under reduced pressure. The obtained oily crude productwas purified by thin layer silica gel chromatography (lower layer ofchloroform:methanol:water=8:3:1→chloroform:methanol=10:1, develop 4times) to obtain the title compound (20 mg, 49%).

¹H-NMR (CD₃OD/CDCl₃) δ: 1.34 (9H, s), 1.73 (2H, brd), 1.90 (3H, m), 2.15(1H, m), 2.70 (6H, s), 3.31-3.44 (6H, m), 3.81 (1H, d, J=14.65Hz), 3.99(1H, m), 4.89 (1H, s), 6.62 (1H, s), 7.23 (1H, d, J=15.38Hz), 7.56 (1H,d, J=6.84Hz), 7.82 (1H, d, J=15.38Hz), 8.06 (1H, s), 9.03 (1H, d,J=6.84Hz).

EI-MS; m/s: 626 (M⁺+1).

(D) [3-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2yl]amino)carbonyl)-3-[(E)-2-carboxy-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2yl}hexahydro-3-pyridinyl)oxy]carbonyl]amino)propyl](trimethyl)ammonium

(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-1[([3-(dimethylamino)propyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid (20 mg, 0.03 mmol) was added with N,N-dimethylformamide (2.5 ml)and methyl iodide (100 μl, 1.2 mmol, 40 eq) and left stand in therefrigerator for 64 hours. After completion of the reaction wasconfirmed, the solvent was concentrated under reduced pressure, and theresulting crude product was purified by liquid chromatography(acetonitrile:water=30:70, 0.1% formic acid solution, flow rate: 9cc/min) to obtain the title compound (17 mg, 83%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.69 (1H, brd), 1.93 (5H, m), 3.15 (9H,8), 3.16 (2H, m), 3.31 (1H, brd), 3.44 (1H, brd), 3.71 (2H, m), 3.93(2H, m), 4.86 (1H, brd), 6.75 (1H, s), 7.03 (1H, brd), 7.59 (2H, brd),7.99 (1H, brd), 8.94 (1H, s).

ES-MS; m/s: 640 (M⁺).

Example 238(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino)carbonyl)-4-oxo-2-{(3R)-3-[({[2-(1-pyridinyl)ethyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl)-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A)(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(2-chloroethyl)amino]carbonyl]oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl)-2-propenoicacid

A crude compound of tert-butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(2-chloroethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(620 mg, 0.94 mmol) was added with 4 N hydrochloric acid in dioxane (30ml) at room temperature and stirred at the same temperature for 11hours. The reaction solution was concentrated under reduced pressure,and the resulting crude product was purified by silica gelchromatography (chloroform→chloroform:methanol=100:1→70:1→50:1→20:1) andfurther purified by HPLC to obtain the title compound (57 mg).

¹H-NMR (CDCl₃) δ: 1.39 (9H, 8), 1.80-2.04 (4H, m), 3.43 (2H, m), 3.51(2H, m), 3.70 (3H, m), 4.03 (1H, brd), 4.90 (1H, brd), 5.29 (1H, brd),6.65 (1H, s), 7.28 (1H, d, J=17.31Hz), 7.76 (2H, m), 8.25 (1H, s), d),9.07 (1H, d, J=7.31Hz).

EI-MS; m/s: 603 (M⁺).

(B)(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-2-{(3R)-3-[({[2-(1-pyridinyl)ethyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl}-4H-pyrido[1,2-a]-pyrimidin-3-yl)-2-propenoicacid

(E)-3-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(2-chloroethyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid (57 mg, 0.10 mmol) was added with pyridine (3 ml) and stirred at100° C. for 17 hours. After completion of the reaction, the solvent wasconcentrated under reduced pressure, and the resulting crude product waspurified by HPLC to obtain the title compound (25 mg, 41%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.69 (1H, m), 1.80 (1H, m), 1.90 (2H,m), 3.46 (1H, m), 3.65 (5H, m), 4.66 (3H, m), 6.75 (1H, s), 7.03 (1H, d,J=15.38Hz), 7.61 (2H, m), 7.96 (1H, s), 8.11 (2H, m), 8.37 (1H, brd),8.61 (1H, t, J=7.81Hz), 8.95 (3H, m).

ES-MS; m/s: 646 (M⁺).

Example 2391-[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-hexahydro-3-pyridinyl}oxy}carbonyl}amino}ethyl}pyridinium

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

Reactions were performed by usingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(400 mg, 0.72 mmol) in the same manner as in Example 3, (A) to obtainthe title compound (500 mg, 100%).

¹H-NMR (CD₃OD/CDCl₃) δ: 1.36 (9H, s), 1.66 (2H, m), 1.93 (2H, m), 3.49(1H, m), 3.64 (1H, m), 3.79 (3H, m), 3.90 (1H, m), 4.03 (1H, m), 4.59(1H, brd), 5.59 (2H, s), 6.64 (1H, s), 6.93 (2H, d, J=8.53Hz), 7.37 (2H,d, J=8.53Hz), 7.62 (1H, brd), 7.70 (1H, d, J=15.60Hz), 7.87 (1H, d,J=15.60Hz), 8.08 (1H, brd), 9.02 (1H, d, J=7.32Hz).

EI-MS; m/s: 642 (M⁺+1).

(B)(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)hexahydro-3-pyridinylN-(2-chloroethyl)carbamate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(500 mg, 0.8 mmol) was added with tetrahydrofuran (8 ml) and pyridine(100 μl, 1.3 mmol, 1.6 eq), then added with 2-chloroethyl isocyanate(150 μl, 1.6 mmol, 2 eq) and stirred at 80° C. for 2 hours. Then themixture was added with 2-chloroethyl isocyanate (550 μl, 5.9 mmol, 7.3eq) and further stirred at 80° C. for 4 hours. The reaction mixture wasreturned to room temperature, added with saturated aqueous sodiumhydrogencarbonate to terminate the reaction and extracted withchloroform. The collected organic layer was dried over magnesiumsulfate, and the solvent was concentrated under reduced pressure. Theresulting crude product was purified by silica gel chromatography(chloroform→chloroform:methanol=80:1→50:1) to obtain the target compound(570 mg) with contained impurities.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.36 (9H, s), 1.73 (1H, m), 1.93 (3H, m), 3.35(3H, m), 3.50 (2H, m), 3.62 (3H, m), 3.80 (3H, s), 4.85 (1H, brd), 5.59(2H, s), 6.63 (1H, s), 6.93 (2H, d, J=8.78Hz), 7.37 (2H, d, J=8.78Hz),7.63 (1H, m), 7.91 (2H, q, J=15.38Hz), 8.12 (1H, brd), 9.02 (2H, d,J=7.57Hz).

EI-MS; m/s: 747 (M⁺).

(C)1-{2-[({[(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)hexahydro-3-pyridinyl]oxy}carbonyl)amino]ethyl}pyridinium

(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-(2-chloroethyl)carbamate (137 mg, 0.18 mmol) was added with pyridine(8 ml) and stirred at 100° C. for 16 hours and 30 minutes. Aftercompletion of the reaction, the solvent was concentrated under reducedpressure, and the resulting crude product was purified by thin layersilica gel column chromatography (chloroform:methanol:water=8:3:1) toobtain the title compound (122 mg, 84%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.67 (1H, m), 1.84 (3H, m), 3.37-3.80(6H, m),3.75 (3H, s), 4.64 (1H, brd), 4.73 (2H, m), 5.57 (2H, d,J=6.12Hz), 6.70 (1H, s), 6.91 (2H, d, J=8.82Hz), 7.32 (2H, d, J=8.82Hz),7.59 (1H, m), 7.78 (2H, m), 7.93 (1H, brd), 8.07 (2H, t, J=7.10Hz), 8.53(1H, t, J=7.10Hz), 8.94 (3H, m).

EI-MS; m/s: 790 (M⁺).

(D)1-[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl]pyridinium

1-{2-[({[(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinyl]oxy}carbonyl)amino]ethyl}pyridinium(122 mg, 0.15 mmol) was added with trifluoroacetic acid (8 ml) at roomtemperature and stirred at the same temperature for 3 hours and 30minutes. As the reaction did not proceed, the reaction mixture washeated to 60° C. and further stirred for 2 hours and 30 minutes. Aftercompletion of the reaction, the solvent was concentrated under reducedpressure, and the resulting crude product was purified by thin layersilica gel column chromatography (lower layer ofchloroform:methanol:water=8:3:1) to obtain the title compound (74 mg,72%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.70 (1H, m), 1.83 (2H, m), 1.97 (1H,m), 3.37-3.50 (2H, m), 3.71 (4H, m), 4.69 (3H, m), 6.75 (1H, s), 7.56(1H, d, J=15.41Hz), 7.61 (1H, d, J=7.35Hz), 8.00 (4H, m), 8.48 (1H, t,J=7.84Hz), 8.83 (2H, d, J=5.63Hz), 9.01 (1H, d, J=7.35Hz).

ES-MS; m/s: 670 (M⁺).

Example 240(2-Amino-2-oxoethyl)[3-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]-amino}carbonyl)-3-[(E)-2-carboxy-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl]dimethylammonium

(A) tert-Butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[({[3-(dimethylamino)propyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoatetert-Butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-[(3R)-3-({[(3-chloropropyl)amino]carbonyl}oxy)hexahydro-1-pyridinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(198 mg, 0.29 mmol) was added with a dimethylamine solution intetrahydrofuran (2.0 M, 5 ml, 10 mmol, 34 eq) and heated to 65° C. Thenthe dimethylamine solution in tetrahydrofuran was occasionally addeduntil completion of the reaction, 15 ml (30 mmol, 103 eq) was used intotal and the reaction was performed for 30 hours. The reaction solutionwas added with saturated brine and extracted with chloroform. Thecollected organic layer was dried over magnesium sulfate. The solventwas concentrated under reduced pressure, and the resulting crude productwas purified by thin layer silica gel chromatography(chloroform:methanol=15:1) to obtain the title compound (32 mg, 16%).

¹H-NMR (CDCl₃) δ: 1.33 (9H, s), 1.52 (9H, s), 1.72 (3H, brd), 1.92 (3H,m), 2.25 (6H, s), 2.41 (2H, m), 3.26 (2H, m), 3.49 (1H, m), 3.67 (2H,m), 3.83 (1H, m), 4.90 (1H, s), 5.84 (1H, brd), 6.60 (1H, s), 7.10 (1H,d, J=15.36Hz), 7.47 (1H, d, J=7.56Hz), 7.57 (1H, d, J=15.36Hz), 7.96(1H, s), 8.98 (1H, d, J=7.56Hz).

EI-MS; m/s: 682 (M⁺+1).

(B)(2-Amino-2-oxoethyl)(3-{[({(3R)-1-[3-[(E)-3-(tert-butoxy)-3-oxo-1-propenyl]-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl]-hexahydro-3-pyridinyl}oxy)carbonyl]amino}propyl)dimethylammonium

tert-Butyl(E)-3-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{(3R)-3-[({[3-(dimethylamino)propyl]amino}carbonyl)oxy]hexahydro-1-pyridinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoate(32 mg, 0.05 mmol) was added with iodoacetamide (15 mg, 0.09 mmol, 1.8eq) and N,N-dimethylformamide (4 ml) and stirred at room temperature for14 hours. After completion of the reaction, the solvent was concentratedunder reduced pressure and the resulting crude product was purified bythin layer silica gel column chromatography(chloroform:methanol:water=6:4:1) to obtain the title compound (48 mg,˜quantitative).

¹H-NMR (CD₃OD) δ: 1.36 (9H, s), 1.54 (9H, s), 1.73 (1H, brd), 2.01 (5H,m), 3.19 (2H, m), 3.62 (2H, m), 3.69 (6H, s), 3.82 (2H, m), 4.14 (2H,m), 4.60 (2H, m), 4.83 (1H, s), 6.66 (1H, d, J=14.67Hz), 6.97 (1H, d,J=15.16Hz), 7.53 (1H, d, J=15.16Hz), 7.62 (1H, s), 8.06 (1H, s), 8.94(1H, s).

EI-MS; m/s: 739 (M⁺).

(C)(2-Amino-2-oxoethyl)[3-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-carboxy-1-ethenyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl]dimethylammonium

Reactions were performed in the same manner as in Example 238, (A) byusing(2-amino-2-oxoethyl)(3-{[({(3R)-1-[3-[(E)-3-(tert-butoxy)-3-oxo-1-propenyl]-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl]hexahydro-3-pyridinyl}oxy)carbonyl]amino}propyl)dimethylammonium(48 mg, 0.06 mmol) and 4 N hydrochloric acid in dioxane (6 ml), and theresulting crude product was purified by thin layer silica gel columnchromatography (lower layer of chloroform:methanol:water=8:3:1) toobtain the title compound (18.5 mg, 49%).

¹H-NMR (CD₃OD/CDCl₃) δ: 1.36 (9H, s), 1.78 (2H, m), 2.03 (4H, m), 3.15(1H, m), 3.25 (1H, m), 3.35 (6H, s), 3.39 (2H, m), 3.68 (2H, m), 3.86(1H, m), 3.95 (1H, m), 4.32 (2H, m), 4.85 (1H, s), 6.64 (1H, s), 7.18(1H, d, J=15.11Hz), 7.61 (1H, d, J=7.56Hz), 7.69 (1H, d, J=15.11Hz),8.08 (1H, s), 8.98 (1H, d, J=7.56Hz).

ES-MS; m/s: 683 (M⁺).

Example 241(2-Amino-2-oxoethyl)[2-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]-amino}-carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino}ethyl}-dimethylammonium

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(7.1 g, 12.7 mmol) was dissolved in dimethylformamide (50 ml) andacetonitrile (200 ml) and cooled to 0° C. with ice, The mixture wasadded dropwise with diphenylphosphonyl chloride (5.3 ml, 25.4 mmol, 2eq) and diisopropylamine (8.8 ml, 50.8 mmol, 4 eq) and stirred at thesame temperature for 15 minutes. Then the mixture was added withdiphenylphosphonyl chloride (1.3 ml, 6.4 mmol, 0.5 eq) anddiisopropylamine (2.5 ml, 12.7 mmol, 1 eq) and further stirred at 0° C.for 10 minutes. The reaction solution was heated to 80° C., added with(R)-(+)-3-hydroxypiperidine hydrochloride (2.6 g, 19 mmol, 1.5 eq) anddiisopropylamine (4.4 ml, 25.4 mmol, 2 eq) and after 30 minutes, addedwith (R)-(+)-3-hydroxypiperidine hydrochloride (3.3 g, 25.4 mmol, 2 eq)and diisopropylamine (9 ml, 50.8 mmol, 4 eq). After further stirred for40 minutes, the reaction solution was returned to room temperature,added with 1 N hydrochloric acid and extracted with chloroform. Theorganic layer was neutralized by adding saturated aqueous sodiumhydrogencarbonate, and the collected organic layer was dried overmagnesium sulfate. The solvent was concentrated under reduced pressure,and the obtained oily substance was purified by silica gel columnchromatography (chloroform→chloroform:ethylacetate=3:1→1:1→chloroform:methanol=50:1→30:1→10:1) to obtain the titlecompound (5.92 g, 73%) as a yellow oily substance with containeddimethylformamide.

¹H-NMR (CDCl₃) δ: 1.38 (9H, s), 1.52 (1H, m), 1.87 (3H, m), 3.50-3.83(4H, m), 3.77 (3H, s), 4.07 (1H, s), 5.50 (2H, s), 6.49 (1H, s), 6.89(2H, d, J=8.53Hz), 7.30 (2H, d, J=8.53Hz), 7.44 (1H, d, J=5.83Hz), 7.78(2H, s), 8.05 (1H, s), 8.83 (1H, s).

EI-MS; m/s: 642 (M⁺+1)

(B)(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-(2-chloroethyl)carbamate

Reactions were performed in the same manner as in Example 239, (B) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(6 g, 9.4 mmol), tetrahydrofuran (70 ml), pyridine (0.75 ml, 9.4 mmol, 1eq) and 2-chloroethyl isocyanate (19 ml, 179 mmol, 19 eq), and theresulting crude product was purified by silica gel chromatography(chloroform→chloroform:ethyl acetate=2:1→1:1) to obtain the titlecompound (7.97 g) with contained impurities.

¹H-NMR (CDCl₃) δ: 1.36 (9H, s), 1.73 (1H, brd), 1.93 (3H, m), 3.35 (2H,m), 3.50 (2H, m), 3.62 (2H, m), 3.80 (3H, s), 3.81 (2H, m), 4.85 (1H,s), 5.59 (2H, s), 6.63 (1H, brd), 6.92 (2H, d, J=8.55Hz), 7.37 (2H, d,J=8.55Hz), 7.63 (1H, brd), 7.91 (2H, dd, J=17.59, 15.38Hz), 8.12 (1H,brd), 9.02 (1H, d, J=7.56Hz).

EI-MS; m/s: 748 (M⁺+1).

(C)(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate

(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-(2-chloroethyl)carbamate (7.97 g, 10.7 mmol) was added with 50%aqueous solution of N,N-dimethylamine (30 ml, 0.53 mol, 50 eq) andethanol (20 ml) and stirred at 80° C. for 5 hours. The reaction solutionwas returned to room temperature, added with saturated brine andextracted with chloroform, and the organic layer was dried overmagnesium sulfate. The solvent was concentrated under reduced pressure,and the resulting crude product was purified by silica gel columnchromatography(chloroform→chloroform:methanol=100:1→70:1→50:1→30:1→10:1) to obtain thetitle compound (3.6 g, 37% for the four steps).

¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 1.68 (1H, brd), 1.96 (3H, m), 2.20 (6H,s), 2.50 (2H, s), 3.33 (3H, m), 3.62 (1H, m), 3.72 (2H, s), 3.77 (3H,s), 4.85 (1H, s), 5.49 (2H, s), 6.11 (1H, brd), 6.55 (1H, s), 6.88 (2H,d, J=8.55Hz), 7.35 (2H, d, J=8.55Hz), 7.46 (1H, d, J=7.32Hz), 7.84 (3H,m), 8.90 (1H, d, J=7.32Hz).

EI-MS; m/s: 756 (M⁺+1).

(D) (2-Amino-2-oxoethyl)[2-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl]dimethylammonium

(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate (3.6 g, 4.8 mmol) was added withiodoacetamide (1 g, 5.3 mmol, 1.1 eq) and dimethylformamide (30 ml) andstirred at room temperature for 17 hours and 30 minutes. Aftercompletion of the reaction, the solvent was concentrated under reducedpressure and used in the following reaction without being purified.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.74-2.17 (4H, m), 3.20 (1H, m), 3.35(1H, m), 3.42 (3H, s), 3.46 (3H, s), 3.67 (3H, s), 3.69-4.04 (6H, m),4.61 (2H, s), 4.82 (1H, s), 5.54 (2H, s), 6.62 (1H, s), 6.91 (2H, d,J=8.82Hz), 7.36 (2H, d, J=8.82Hz), 7.55 (1H, d, J=7.35Hz), 7.97 (3H, m),9.00 (1H, d, J=7.35Hz).

EI-MS; m/s: 813 (M⁺).

(E)(2-Amino-2-oxoethyl)[2-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}-carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl]dimethylammonium

(2-Amino-2-oxoethyl)[2-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl]dimethylammonium(4.48 g, 4.8 mmol) was added with anisole (5 ml) and trifluoroaceticacid (160 ml) and stirred at 60° C. for 3 hours. After completion of thereaction, the solvent was concentrated under reduced pressure, and thecrude product was added with diisopropyl ether. The precipitated solidwas taken by filtration and washed with diisopropylether to remove mostof anisole. The resulting solid was purified by liquid chromatography(methanol:0.1% acetic acid aqueous solution=60:40, flow rate: 25 cc/min)to obtain the title compound (825 mg, 25%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.71 (1H, m), 1.98 (3H, m), 3.32 (6H,s), 3.63 (6H, m), 3.89 (2H, m), 4.17 (2H, m), 4.80 (1H, m), 6.73 (1H,s), 7.52 (1H, d, J=16.14Hz), 7.57 (1H, d, J=7.34Hz), 7.94 (1H, d,J=16.14Hz), 8.00 (1H, s), 8.96 (1H, d, J=7.34Hz).

FAB-MS; m/s: 693 (M⁺).

Example 242(E)-3-[2-{3-[(Aminocarbonyl)oxy]piperidino}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A) tert-Butyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

Reactions were performed in the same manner as in Example 231, (C) byusing tert-butyl(E)-3-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoate(695 mg, 1.5 mmol), and the resulting crude product was purified bysilica gel column chromatography(chloroform→chloroform:methanol=100:1→80:1→60:1) to obtain the titlecompound (709 mg) with contained impurities.

¹H-NMR (CDCl₃) δ: 1.30 (9H, s), 1.38 (9H, s), 1.72 (2H, m), 1.82 (2H,m), 2.91 (2H, m), 3.53 (2H, m), 3.60 (1H, m), 6.57 (1H, s), 7.94 (1H,s), 8.90 (1H, m).

EI-MS; m/s: 554 (M⁺+1).

(B) tert-Butyl(E)-3-[2-{3-[(aminocarbonyl)oxy]piperidino}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate

Reactions were performed in the same manner as in Example 232, (D) byusing tert-Butyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(3-hydroxypiperidino)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(709 mg, 1.28 mmol), and the resulting crude product was purified bysilica gel column chromatography (chloroform→chloroform:methanol=100:1)to obtain the title compound (670 mg) with contained impurities.

¹H-NMR (CDCl₃) δ: 1.30 (9H, s), 1.51 (9H, s), 1.48 (1H, m), 1.75 (3H,m), 3.24 (3H, m), 3.42 (1H, m), 4.51 (1H, brd), 6.60 (1H, s), 7.11 (1H,d, J=13.16Hz), 7.45 (1H, dd, J=7.56, 4.84Hz), 7.53 (1H, d, J=13.16Hz),8.07 (1H, s), 8.95 (1H, d, J=7.56Hz).

(C)(E)-3-[2-{3-[(Aminocarbonyl)oxy]piperidino}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

Reactions were performed in the same manner as in Example 232, (E) byusing tert-Butyl(E)-3-[2-[3-[(aminocarbonyl)oxy]piperidino}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate (670 mg, 1.12 mmol), and theresulting crude product was purified by silica gel column chromatography(chloroform→chloroform:methanol=100:1→80:1→50:1→10:1→chloroform:methanol:water=6:4:1)to obtain the title compound (40 mg, 5% for the three steps).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.70 (1H, m), 1.85 (1H, m), 2.00 (2H,m), 3.56 (1H, m), 3.71 (1H, m), 3.84 (2H, m), 4.86 (1H, m), 6.74 (1H,s), 7.00 (1H, d, J=15.63Hz), 7.56 (1H, dd, J=7.32, 1.71Hz), 7.61 (1H, d,J=15.63Hz), 8.01 (1H, s), 8.92 (1H, d, J=7.32Hz).

ES-MS; m/s: 541 (M⁺+1).

Example 243N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[4-({2-[1-(2-amino-2-oxoethyl)-1,1-dimethylammonio]acetyl}amino)piperidino]-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2a]pyrimidine -8-carboxyamide

(A) N-(1-Benzyl-piperidin-4-yl)-2-(dimethylamino)acetamide

1-Benzyl-4-piperidinamine (3 g, 16 mmol) was added with dichloromethane(30 ml), dimethylaminoacetic acid (1.95 g, 19 mmol),1-hydroxybenzotriazole (2.6 g, 19.2 mmol, 1.2 eq) and triethylamine (3.3ml, 24 mmol, 1.5 eq) at room temperature, then added with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (4.5 g, 24mmol, 1.5 eq) at room temperature and stirred at the same temperaturefor 16 hours and 30 minutes. After completion of the reaction, themixture was added with saturated aqueous sodium hydrogencarbonate andextracted with chloroform. The organic layer was washed with saturatedbrine, and the collected organic layer was dried over magnesium sulfate.The solvent was concentrated under reduced pressure, and the resultingcrude product was purified by silica gel column chromatography(chloroform→chloroform:methanol=100:1→80:1→50:1→30:1→10:1) to obtain thetitle compound (4.6 g, 100%).

¹H-NMR (CDCl₃) δ: 1.50 (2H, dq, J=11.23, 3.91Hz), 1.89 (2H, dd, J=12.45,3.91Hz), 2.13 (2H, d, J=11.23Hz), 2.27 (6H, s), 2.80 (2H, d, J=12.45Hz),2.91 (2H, s), 3.49 (2H, s), 3.82 (1H, m), 7.04 (1H, d, J=7.81Hz), 7.27(5H, m).

EI/MS; m/z: 276 (M⁺+1).

(B) N¹-(4-Piperidyl)-2-(dimethylamino)acetamide

N-(1-Benzyl-piperidin-4-yl)-2-(dimethylamino)acetamide (4.6 g, 17 mmol)was added with methanol (16 ml) and 20% palladium hydroxide carbon (500mg), attached with a balloon containing hydrogen, and stirred at roomtemperature for 16 hours. After completion of the reaction, palladiumwas removed by filtration through a Celite layer, and the solvent wasconcentrated under reduced pressure to obtain the title compound (3.4 g,100%) without purification.

¹H-NMR (CD₃OD) δ: 1.77 (2H, q, J=11.94Hz), 2.06 (2H, d, J=11.94Hz), 2.34(6H, s), 3.07 (4H, m), 3.34 (2H, m), 3.97 (1H, m).

EI/MS; m/z: 186 (M⁺+1).

(C)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-(4-{[2-(dimethylamino)acetyl]amino}-piperidino)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(310 mg, 0.6 mmol) was dissolved in dimethylformamide (6.5 ml) andacetonitrile (11 ml) and cooled to −10° C. with ice. The mixture wasadded dropwise with diphenylphosphonyl chloride (0.23 ml, 1.2 mmol, 2eq) and diisopropylamine (0.38 ml, 2.4 mmol, 4 eq) and stirred at thesame temperature for 5 minutes. Then the mixture was added withdiphenylphosphonyl chloride (0.23 ml, 1.2 mmol, 2 eq) anddiisopropylamine (0.38 ml, 2.4 mmol, 4 eq) and further stirred at −10°C. for 15 minutes. The reaction mixture was added with2-dimethylamino-N-piperidin-4-yl-acetamide (155 mg, 0.9 mmol, 1.5 eq),heated to 80° C., stirred for 1 hour and then added with2-dimethylamino-N-piperidin-4-yl-acetamide (200 mg, 1.2 mmol, 2 eq) anddiisopropylamine (0.3 ml, 1.8 mmol, 3 eq). After further stirred for 40minutes, the reaction solution was returned to room temperature, addedwith water and extracted with chloroform, and the collected organiclayer was dried over magnesium sulfate. The solvent was concentratedunder reduced pressure, and the obtained oily substance was purified bysilica gel column chromatography(chloroform→chloroform:methanol=100:1→70:1→40:1→10:1) and furtherpurified by thin layer silica gel column chromatography(chloroform:methanol=20:1) to obtain the title compound (182 mg, 45%).

¹H-NMR (CDCl₃) δ: 1.37 (9H, s), 1.59 (1H, m), 1.86 (2H, d, J=13.23Hz),2.00 (1H, m), 2.26 (6H, 8), 2.92 (2H, s), 3.08 (1H, m), 3.11 (1H, t,J=12.23Hz), 3.73 (2H, m), 3.78 (3H, 8), 3.89 (1H, m), 4.04 (1H, m), 5.53(2H, a), 6.57 (1H, s), 6.90 (2H, d, J=7.56Hz), 7.16 (2H, d, J=7.56Hz),7.45 (1H, d, J=7.09Hz), 7.75 (1H, d, J=15.41Hz), 7.92 (1H, d,J=15.41Hz), 7.96 (1H, s), 8.88 (1H, d, J=7.09Hz).

EI-MS; m/s: 726 (M⁺+1).

(D)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[4-({2-[1-(2-amino-2-oxoethyl)-1,1-dimethylammonio]acetyl)amino)piperidino]-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

Reactions were performed in the same manner as in Example 11, (D) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-(4-{[2-(dimethylamino)acetyl]aminolpiperidino)3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-l-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide (182 mg, 0.25mmol), and the resulting crude product was purified by thin layer silicagel column chromatography (lower layer ofchloroform:methanol:water=8:3:1) to obtain the title compound (129 mg,66%).

¹H-NMR (CDCl₃) δ: 1.30 (9H, s), 1.82 (1H, m), 2.06 (3H, m), 2.90 (2F,m), 3.28 (2H, m), 3.67 (3H, s), 3.73 (6H, s), 4.70 (5H, m), 5.48 (2H,s), 6.56 (1H, s), 6.85 (2H, d, J=8.79Hz), 7.18 (2H, d, J=8.79Hz), 7.47(1H, s), 7.54 (1H, d, J=15.87Hz), 7.78 (1H, d, J=15.87Hz), 7.87 (1H, s),8.85 (1H, s).

EI-MS; m/s: 783 (M⁺).

(E)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[4-({2-[1-(2-amino-2-oxoethyl)-1,1-dimethylammonio]acetyl}amino)piperidino]-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2a]pyrimidine-8-carboxyamide

Reactions were performed in the same manner as in Example 9, (D) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-[4-({2-[1-(2-amino-2-oxoethyl)-1,1-dimethylammonio]acetyl}amino)piperidino]-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide (129 mg, 0.16 mmol), and the resulting crude product waspurified by thin layer silica gel column chromatography (lower layer ofchloroform:methanol:water=8:3:1) to obtain the title compound (28 mg,26%).

¹H-NMR (CD₃OD/CDCl₃) δ: 1.35 (9H, s), 1.70 (2H, m), 1.97 (2H, m), 3.09(2H, t, J=12.21Hz), 3.41 (2H, m), 3.50 (6H, s), 4.40 (5H, m), 6.60 (1H,s), 7.36 (1H, d, J=16.11Hz), 7.53 (1H, d, J=7.57Hz), 7.89 (1H, d,J=16.11Hz), 7.99 (1H, s), 8.94 (1H, d, J=7.57Hz).

FAB-MS; m/s: 663 (M⁺).

Example 244[3-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl](trimethyl)ammonium

(A) tert-Butyl (3R)-3-hydroxyhexahydro-1-pyridinecarboxylate

(3R)-(+)-Hydroxypiperidine hydrochloride (1 g, 7.3 mmol) was added withdichloromethane (15 ml) and methanol (10 ml) and cooled to 0° C. withice. The reaction mixture was added with triethylamine (2.5 ml, 18.3mmol, 2.5 eq) and di-tert-butyl dicarbonate (2.4 g, 11 mmol, 1.5 eq) andstirred at the same temperature for 1 hour. The mixture was added withwater to terminate the reaction and extracted with chloroform, and thecollected organic layer was dried over magnesium sulfate. The solventwas concentrated under reduced pressure, and the resulting crude productwas purified by silica gel column chromatography to obtain the titlecompound (1.3 g, 89%).

¹H-NMR (CDCl₃) δ: 1.45 (9H, s), 1.46 (2H, m), 1.75 (1H, m), 1.89 (1H,brd), 3.02 (2H, m), 3.57 (1H, brd), 3.71 (1H, brd), 3.78 (1H, dd,J=12.70, 3.91Hz).

(B) tert-Butyl(3R)-3-({([(3-chloropropyl)amino]carbonyl}oxy)hexahydro-1-pyridinecarboxylate

Reactions were performed in the same manner as in Example 7, (A) byusing tert-butyl (3R)-3-hydroxyhexahydro-1-pyridinecarboxylate (730 mg,3.6 mmol), and the resulting crude product was purified by silica gelcolumn chromatography (chloroform→chloroform:methanol=100:1→80:1→50:1)to obtain the title compound (1.14 g, 98%).

¹H-NMR (CDCl₃) δ: 1.45 (9H, s), 1.69-1.99 (6H, m), 3.34-3.67 (8H, m),4.67 (1H, brd).

(C) tert-Butyl(3R)-3-[({[3-(dimethylamino)propyl]amino}carbonyl)oxy]hexahydro-1-pyridinecarboxylate

tert-Butyl(3R)-3-({[(3-chloropropyl)amino]carbonyl}oxy)hexahydro-1-pyridinecarboxylate(100 mg, 0.3 mmol) was added with 50% aqueous solution of dimethylamine(2 ml) and ethanol (1 ml) and stirred in a sealed tube at 130° C. for 4hours. After completion of the reaction, the reaction mixture wasextracted with chloroform, and the collected organic layer was driedover magnesium sulfate. The solvent was concentrated under reducedpressure to obtain the title compound.

¹H-NMR (CDCl₃) δ: 1.44 (9H, s), 1.73 (2H, brd), 1.85 (3H, brd), 2.08(1H, brd), 2.65 (4H, brd), 3.29 (6H, s), 3.48 (4H, brd), 4.66 (1H, brd).

EI-MS; m/s: 330 (M⁺+1).

(D) (3R)-Hexahydro-3-pyridinyl N-[3-(dimethylamino)propyl]carbamate

tert-Butyl(3R)-3-[({[3-(dimethylamino)propyl]amino}carbonyl)oxy]hexahydro-1-pyridinecarboxylate(885 mg, 2.69 mmol) was added with 4 N hydrochloric acid in dioxane (30ml) at room temperature and stirred at the same temperature for 2 hours.After completion of the reaction, the solvent was concentrated underreduced pressure to obtain the title compound (730 mg) without purifyingthe obtained product.

¹H-NMR (CD₃OD) δ: 1.97 (6H, brd), 2.90 (3H, s), 2.91 (3H, s), 2.97 (1H,m), 3.07 (1H, m), 3.30 (4H, m), 3.66 (2H, m), 5.01 (1H, brd).

EI-MS; m/s: 230 (M⁺+1).

(E)(3R)-1-(8-([4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)hexahydro-3-pyridinylN-[3-(dimethylamino)propyl]carbamate

Reactions were performed in the same manner as in Example 3, (A) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[2-(4-methoxybenzyl)-21H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-6oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(250 mg, 0.45 mmol) and (3R)-hexahydro-3-pyridinylN-[3-(dimethylamino)propyl]carbamate (540 mg, 1.83 mmol, 4 eq), and theresulting crude product was purified by silica gel column chromatography(chloroform:methanol=10:1) to obtain the title compound (357 mg, 100%)with contained impurities.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.65 (1H, brd), 1.75 (1H, brd), 1.97(3H, brd), 2.11 (1H, brd), 2.58 (6H, s), 2.90 (2H, m), 3.28 (2H, m),3.72 (4H, m), 3.77 (3H, m), 4.80 (1H, brd), 5.50 (2H, s), 6.58 (1H, s),6.88 (2H, d, J=8.53Hz), 7.34 (2H, d, J=8.53Hz), 7.49 (1H, d, J=7.56Hz),7.96 (3H, d, J=11.94Hz), 8.96 (2H, d, J=7.56Hz).

EI-MS; m/s: 770 (M⁺+1).

(F)[3-({[((3R)-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxylcarbonyl}amino)propyl](trimethyl)ammonium

Reactions were performed in the same manner as in Example 6, (D) byusing(3R)-1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[3-(dimethylamino)propyl]carbamate (253 mg, 0.33 mmol), and theresulting crude product was purified by thin layer silica gel columnchromatography (lower layer of chloroform:methanol:water=8:3:1) toobtain the title compound (71 mg).

¹H-NMR (CD₃OD/CDCl₃) δ: 1.36 (9H, s), 1.71 (1H, brd), 1.99 (5H, brd),3.13 (9H, s), 3.19-3.38 (6H, m), 3.70 (2H, m), 3.78 (3H, s), 4.91 (1H,brd), 5.61 (2H, s), 6.72 (1H, s), 6.92 (2H, m), 7.34 (2H, m), 7.87 (3H,m), 8.06 (1H, s), 9.01 (1H, s).

EI-MS; m/s: 784 (M⁺).

(G)[3-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl](trimethyl)ammonium

Reactions were performed in the same manner as in Example 239, (D) byusing[3-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl](trimethyl)ammonium (71 mg, 0.09 mmol), and the resulting crude product waspurified by thin layer silica gel column chromatography(chloroform:methanol:water=6:4:1) to obtain the title compound (19 mg,32%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.69 (1H, brd), 1.92 (4H, brd), 2.05(1H, brd), 3.11 (9H, s), 3.12-3.34 (5H, m), 3.53 (1H, d, J=13.92Hz),3.94 (2H, m), 4.86 (1H, brd), 6.75 (1H, s), 7.93 (2H, m), 7.95 (1H, d,J=15.38Hz), 8.04 (1H, s), 8.98 (1H, d, J=7.08Hz).

FAB-MS; m/s: 664 (M⁺).

Example 245(2-Amino-2-oxoethyl)[3-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]-amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido-[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl]-dimethylammonium

(A) (2-Amino-2-oxoethyl) [3-({[((3R)-1-{8-({[4-(tert-butyl)-l,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl]dimethylammonium

Reactions were performed in the same manner as in Example 240, (B) byusing(3R)-1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[3-(dimethylamino)propyl]carbamate (166 mg, 0.22 mmol), and theresulting crude product was purified by thin layer silica gel columnchromatography (lower layer of chloroform:methanol:water=8:3:1) toobtain the title compound (137 mg) with contained impurities.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.40 (9H, s), 1.75 (2H, brd), 2.10 (4H, brd),3.14 (4H, m), 3.30 (3H, s), 3.32 (3H, s), 3.45-3.90 (4H, m), 3.77 (3H,s), 4.26 (2H, s), 4.83 (1H, s), 5.56 (2H, s), 6.63 (1H, s), 6.92 (2H, d,J=8.79Hz), 7.27 (2H, d, J=8.79Hz), 7.64 (1H, d, J=7.32Hz), 7.99 (2H, s),8.11 (1H, s), 9.04 (1H, d, J=7.32Hz).

EI-MS; m/s: 827 (M⁺).

(B) (2-Amino-2-oxoethyl)[3-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl]dimethylammonium

Reactions were performed in the same manner as in Example 239, (D) byusing(2-amino-2-oxoethyl)[3-([((3R)-1-{8-([4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl-hexahydro-3-pyridinyl)oxy]carbonylamino)propyl]dimethylammonium(137 mg, 0.17 mmol), and the resulting crude product was purified bythin layer silica gel column chromatography (lower layer ofchloroform:methanol:water=8:3:1) to obtain the title compound (12 mg,10%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.74 (2H, brd), 1.99 (3H, brd), 2.17(1H, brd), 3.14 (3H, d, J=12.70Hz), 3.22 (3H, s), 3.32 (3H, s), 3.37(1H, m), 3.50 (2H, dd, J=5.75, 6.84Hz), 3.95 (2H, m), 4.15 (2H, dd,J=43.46, 15.63Hz), 4.82 (1H, s), 6.63 (1H, s), 7.59 (1H, d, J=7.81Hz),7.75 (1H, d, J=16.11Hz), 8.08 (1H, d, J=16.11Hz), 8.09 (1H, s), 9.05(1H, d, J=7.81Hz).

FAB-MS; m/s: 707 (M⁺).

Example 246[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](trimethyl)ammonium

(A)(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate

(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-(2-chloroethyl)carbamate (400 mg, 0.54 mmol) was added withN,N-dimethylamine solution in tetrahydrofuran (2.0 M, 60 ml) and heatedto 100° C. in a sealed tube with stirring for 12 hours. After completionof the reaction, the reaction solution was returned to room temperature.The solvent was concentrated under reduced pressure, and the resultingcrude product was purified by thin layer silica gel columnchromatography (chloroform:methanol=10:1) to obtain the title compound(190 mg, 47%).

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.65 (1H, brd), 1.92 (3H, brd), 2.25(6H, s), 2.52 (2H, t, J=6.59Hz), 3.33 (2H, brd), 3.73 (4H, q, J=7.08Hz),3.78 (3H, s), 4.86 (1H, s), 5.51 (2H, s), 6.04 (1H, brd),6.59 (1H, s),6.89 (2H, d, J=8.79Hz), 7.35 (2H, d, J=8.79Hz), 7.49 (1H, d, J=7.32Hz),7.88 (2H, q, J=11.74Hz), 7.92 (1H, s), 8.96 (1H, d, J=7.32Hz).

EI-MS; m/s: 756 (M⁺+1).

(B)[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](trimethyl)ammonium

Reactions were performed in the same manner as in Example 237, (D) byusing(3R)-1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate (90 mg, 0.12 mmol), and theresulting crude product was purified by thin layer silica gel columnchromatography (lower layer of chloroform:methanol:water=8:3:1) toobtain the title compound (87 mg, 95%).

¹H-NMR (CDCl₃) δ: 1.36 (9H, s), 1.67 (1H, brd), 1.81 (2H, brd), 2.14(1H, brd), 3.15 (1H, brd), 3.33 (1H, d, J=12.94Hz), 3.50 (9H, s), 3.73(4H, q, J=7.08Hz), 3.78 (3H, s), 3.89 (2H, brd), 4.78 (1H, s), 5.51 (2H,s), 6.60 (1H, s), 6.91 (2H, d, J=8.55Hz), 7.36 (2H, d, J=8.55Hz), 7.47(1H, brd), 7.89 (1H, s), 7.99 (2H, s), 8.95 (1H, brd).

EI-MS; m/s: 770 (M⁺).

(C)[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](trimethyl)ammonium

Reactions were performed in the same manner as in Example 239, (D) byusing[2-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](trimethyl)ammonium(87 mg, 0.11 mmol), and the resulting crude product was purified by thinlayer silica gel column chromatography (lower layer ofchloroform:methanol:water=8:3:1) to obtain the title compound (52 mg,71%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.70 (1H, brd), 1.97 (3H, brd), 3.16(9H, s), 3.42 (3H, m), 3.55 (3H, m), 3.93 (2H, m), 4.89 (1H, s), 6.75(1H, s), 7.56 (1H, d, J=16.14Hz), 7.60 (1H, dd, J=7.34, 1.22Hz), 7.96(1H, d, J=16.14Hz), 8.03 (1H, s), 8.99 (1H, d, J=7.34Hz).

EI-MS; m/s: 650 (M⁺).

Example 247[2-({[((3S)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl}oxy}carbonyl}amino}ethyl}(trimethyl)ammonium

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3S)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido-[1,2-a]pyrimidine-8-carboxyamide

Reactions were performed in the same manner as in Example 243, (C) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(1 g, 1.8 mmol) and (S)-(−)-3-hydroxypiperidine hydrochloride (1.1 g,8.1 mmol, 4.5 eq), and the resulting crude product was purified bysilica gel column chromatography(chloroform→chloroform:methanol=100:1→80:1→50:1) to obtain the titlecompound (1.3 g, 100%) with contained dimethylformamide.

¹H-NMR (CDCl₃) δ: 1.37 (9H, s), 1.48 (2H, m), 1.84 (2H, m), 3.32 (1H,m), 3.54 (2H, m), 3.64 (1H, m), 3.78 (3H, s), 4.07 (1H, s), 5.51 (2H,s), 6.59 (1H, s), 6.88 (2H, d, J=8.80Hz), 7.33 (2H, d, J=8.80Hz), 7.47(1H, d, J=7.20Hz), 7.78 (2H, s), 8.00 (1H, brd), 8.86 (1H, d, J=7.20Hz).

EI-MS; m/s: 642 (M⁺+1)

(B)(3S)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl}hexahydro-3-pyridinylN-(2-chloroethyl)carbamate

Reactions were performed in the same manner as in Example 9, (B) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-[(3S)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(1.3 g, 2.0 mmol), and the resulting crude product was purified bysilica gel chromatography to obtain the title compound (1.03 g, 68%).

¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 1.73 (1H, brd), 1.93 (3H, m), 3.35 (2H,m), 3.50 (2H, m), 3.60 (2H, m), 3.62 (2H, m), 3.78 (3H, s), 4.83 (1H,s), 5.51 (2H, s), 6.60 (1H, s), 6.88 (2H, d, J=8.57Hz), 7.34 (2H, d,J=8.57Hz), 7.52 (1H, d, J=7.35Hz), 7.96 (1H, s), 7.99 (2H, d, J=7.10Hz),9.00 (1H, d, J=7.35Hz).

EI-MS; m/s: 747 (M⁺).

(C)(3S)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate

Reactions were performed in the same manner as in Example 246, (A) byusing(3S)-1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-(2-chloroethyl)carbamate (1.03 g, 1.4 mmol), and the resulting crudeproduct was purified by silica gel column chromatography to obtain thetitle compound (580 mg, 43% for the three steps).

¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 1.65 (1H, m), 1.89 (3H, m), 2.21 (6H,s), 2.50 (2H, m), 3.32 (2H, m), 3.66 (4H, m), 3.77 (3H, s), 4.85 (1H,brd), 5.50 (2H, s), 6.10 (1H, brd), 6.88 (1H, s), 7.28 (2H, d,J=8.79Hz), 7.34 (2H, d, J=8.79Hz), 7.47 (1H, d, J=7.32Hz), 7.85 (3H, m),8.92 (1H, d, J=7.32Hz).

EI-MS; m/s: 756 (M⁺+1).

(D)[2-({[((3S)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](trimethyl)ammonium

Reactions were performed in the same manner as in Example 237, (D) byusing(3S)-1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate (261 mg, 0.35 mmol), and theresulting crude product was purified by thin layer silica gel columnchromatography (lower layer of chloroform:methanol:water=8:3:1) toobtain the title compound (236 mg, 89%).

¹H-NMR (CD₃OD/CDCl₃) δ: 1.35 (9H, s), 1.74-2.18 (4H, m), 3.23-3.47 (2H,brd), 3.47 (9H, s), 3.68-3.92 (6H, m), 3.43 (3H, s), 4.81 (1H, brd),5.54 (2H, s), 6.61 (1H, s), 6.91 (2H, d, J=8.82Hz), 7.36 (2H, d,J=8.82Hz), 7.56 (1H, dd, J=7.35, 1.96Hz), 8.00 (3H, brd), 9.02 (1H, d,J=7.35Hz).

EI-MS; m/s: 770 (M⁺).

(E)[2-({[((3S)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](trimethyl)ammonium

Reactions were performed in the same manner as in Example 239, (D) byusing[2-({[((3S)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonylamino)ethyl](trimethyl)ammonium(236 mg, 0.31 mmol), and the resulting crude product was purified bythin layer silica gel column chromatography (lower layer ofchloroform:methanol:water=8:3:1) to obtain the title compound (196 mg,98%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.69 (1H, brd), 1.92 (3H, brd), 3.18(9H, s), 3.31 (2H, brd), 3.43 (2H, brd), 3.56 (2H, brd), 3.85 (2H, brd),4.80 (1H, brd), 6.71 (1H, s), 7.49 (1H, d, J=16.36Hz), 7.55 (1H, brd),7.90 (1H, d, J=16.36Hz), 7.95 (1H, brd),8.92 (1H, brd).

ES-MS; m/s: 649 (M⁺).

Example 248(2-Amino-2-oxoethyl])[2-({[((3S)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]-amino}-carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido-[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl]dimethylammonium

(A) (2-Amino-2-oxoethyl)[2-({[((3S)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl]dimethylammonium

Reactions were performed in the same manner as in Example 241, (D) byusing(3S)-1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate (320 mg, 0.42 mmol), and theresultant was used in the following reaction without being purified.

¹H-NMR (CD₃OD/CDCl₃) δ: 1.34 (9H, s), 1.76 (1H, brd), 2.09 (3H, brd),3.20-3.35 (2H, m), 3.41 (3H, s), 3.46 (3H, s), 3.69-3.93 (6H, m), 3.79(3H, s), 4.61 (2H, s), 4.84 (1H, brd), 5.54 (2H, s), 6.62 (1H, s), 6.90(1H, d, J=8.57Hz), 7.36 (1H, d, J=8.57Hz), 7.56 (1H, dd, J=7.35,1.72Hz), 8.01 (3H, m), 9.02 (1H, d, J=7.35Hz).

EI-MS; m/s: 813 (M⁺).

(B)(2-Amino-2-oxoethyll)[2-({[((3S)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}-carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-2-yl}hexahydro-3-pyridinyl)oxylcarbonyl}amino)ethyl]dimethylammonium

Reactions were performed in the same manner as in Example 239, (D) byusing(2-amino-2-oxoethyl)[2-({[((3S)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)-ethyl]dimethylammonium(316 mg, 0.4 mmol), and the resulting crude product was purified by thinlayer silica gel column chromatography (lower layer ofchloroform:methanol:water=8:3:1) to obtain the title compound (195 mg,66% for the two steps).

¹H-NMR (CD₃OD) δ: 1.34 (9H, s), 1.68 (1H, brd), 1.92 (3H, brd), 3.34(6H, s), 3.41 (1H, m), 3.55 (3H, m), 3.66 (2H, m), 3.86 (2H, m), 4.16(2H, s), 4.86 (1H, brd), 6.72 (1H, s), 7.51 (1H, d, J=16.16Hz), 7.56(1H, d, J=7.35Hz), 7.92 (1H, d, J=16.16Hz), 8.99 (1H, s), 8.95 (1H, d,J=7.35Hz).

ES-MS; m/s: 693 (M⁺)

Example 2492-{1-[2-({([(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-4-piperidyl}oxy}carbonyl}amino}ethyl}-1,1-dimethylammonio}acetate

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-(4-hydroxypiperidino)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidine-8-carboxyamide

Reactions were performed in the same manner as in Example 241, (A) byusingN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(500 mg, 0.9 mmol) and 4-hydroxypiperidine (5 mmol, 5.5 eq), and theobtained oily substance was purified by silica gel column chromatography(chloroform→chloroform:ethylacetate=3:1→1:1→chloroform:methanol=80:1→50:1→30:1→10:1) to obtain thetitle compound (578 mg, 100%) as a yellow oily substance with containeddimethylformamide.

¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 1.41 (2H, brd), 1.81 (2H, brd), 3.00(1H, brd), 3.49 (1H, m), 3.63 (1H, m), 3.78 (3H, s), 4.00 (1H, brd),4.16 (1H, brd), 5.53 (2H, s), 6.59 (1H, s), 6.89 (2H, d, J=8.55Hz), 7.37(2H, d, J=8.55Hz), 7.50 (1H, d, J=7.32Hz), 7.74 (1H, d, J=15.38Hz), 7.89(1H, s), 7.91 (1H, d, J=15.38Hz), 8.95 (1H, d, J=7.32Hz).

EI-MS; m/s: 642 (M⁺+1).

(B)1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)-4-piperidyl-N-[2-(dimethylamino)ethyl]carbamate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-(4-hydroxypiperidino)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(578 mg, 0.9 mmol) was added with dichloromethane (9 ml),N,N-dimethylethylenediamine (0.4 ml, 3.6 mmol, 4 eq) and1,1′-carbonyl-bis-1H-imidazole (220 mg, 1.4 mmol, 1.5 eq) at roomtemperature and stirred at the same temperature for 1 hour. The reactionmixture was added with 1,1′-carbonyl-bis-1H-imidazole (100 mg, 0.7 mmol,0.75 eq), after 30 minutes, further added withN,N-dimethylethylenediamine (0.4 ml, 3.6 mmol, 4 eq) and stirred for 15hours. After completion of the reaction, the reaction mixture was addedwith 1 N hydrochloric acid and extracted with chloroform:methanol=10:1.The organic layer was neutralized by adding saturated aqueous sodiumhydrogencarbonate, and the collected organic layer was dried overmagnesium sulfate. The solvent was concentrated under reduced pressure,and the resulting crude product was purified by silica gel columnchromatography (chloroform→chloroform:methanol=60:1→30:1→10:1) to obtainthe title compound (354 mg, 52% for the two steps).

¹H-NMR (CDCl₃) δ: 1.40 (9H, s), 1.53 (1H, brd), 1.69 (1H, brd), 1.78(1H, brd), 1.91 (1H, brd), 2.22 (6H, s), 2.42 (2H, m), 3.14 (1H, brd),3.26 (2H, m), 3.39 (1H, brd), 3.51 (1H, brd), 3.74 (1H, m), 3.79 (3H,s), 4.85 (1H, brd), 5.35 (1H, brd), 5.53 (2H, s), 6.57 (1H, s), 6.92(2H, d, J=8.56Hz), 7.23 (2H, d, J=8.56Hz), 7.38 (1H, d, J=8.56Hz), 7.76(1H, d, J=15.41Hz), 7.82 (1H, s), 7.91 (1H, d, J=15.41Hz), 8.82 (1H,brd).

EI-MS; m/s: 756 (M⁺+1).

(C)2-(1-{2-[({[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-2-yl)-4-piperidyl]oxy}carbonyl)amino]ethyl}-1,1-dimethylammonio)acetate

1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4-piperidyl-N-[2-(dimethylamino)ethyl]carbamate(495 mg, 0.7 mmol) was added with N,N-dimethylformamide (10 ml) andbromoacetic acid tert-butyl ester (106 μl, 0.8 mmol, 1.1 eq), stirred atroom temperature for 2 hours, further added with bromoacetic acidtert-butyl ester (50 μl, 0.4 mmol, 0.5 eq) and further stirred at thesame temperature for 1 hour. After completion of the reaction, thesolvent was evaporated under reduced pressure, and the residue was addedwith 4 N hydrochloric acid in dioxane (10 ml) and stirred at roomtemperature for 14 hours and 30 minutes. After the solvent wasconcentrated under reduced pressure, the reaction mixture was used inthe following reaction without being purified.

ES-MS; m/s: 814 (M⁺).

(D)2-{1-[2-({[(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-4-piperidyl)oxy]carbonyl}amino)ethyl]-1,1-dimethylammonio}acetate

2-(1-{2-[({[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[2-(4-methoxybenzyl)-2H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4-piperidyl]oxy}carbonyl)amino]ethyl}-1,1-dimethylammonio)acetate(557 mg, 0.7 mmol) was added with trifluoroacetic acid (25 ml) andanisole (50 μl) and heated at 60° C. with stirring for 3 hours. Aftercompletion of the reaction, the solvent was concentrated under reducedpressure, and the resulting crude product was purified by liquidchromatography (methanol:water=60:40, flow rate: 10 cc/min) to obtainthe title compound (122 mg, 27% for the three steps).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.87 (2H, brd), 2.07 (2H, brd), 3.29(6H, s), 3.57 (4H, m), 3.73 (2H, m), 3.87 (4H, m), 4.91 (1H, brd), 6.70(1H, s), 7.40 (1H, d, J=16.11Hz), 7.64 (1H, dd, J=7.32, 1.71Hz), 7.93(1H, d, J=16.11Hz), 8.04 (1H, s), 8.96 (1H, d, J=7.32Hz).

ES-MS; m/s: 694 (M⁺+1).

Example 250(E)-3-[2-{4-[2-(2-Aminothiazol-5-yl)-3-pyridin-4-yl-acryloyl]piperazin-1-yl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl]-2-propenoicacid

(A)(E)-2-(2-tert-Butoxycarbonylaminothiazol-5-yl)-3-pyridin-4-yl-2-propenoicacid

A solution of(E)-2-(2-tert-butoxycarbonylaminothiazol-5-yl)-3-pyridin-4-yl-2-propenoicacid ethyl ester (1.13 g, 3.02 mmol) in tetrahydrofuran (6 ml), methanol(2 mL) and water (2 mL) was added with lithium hydroxide monohydrate(190 mg, 4.53 mmol) with ice cooling and stirred at room temperature for21 hours, at 50° C. for 8 hours and at 80° C. for 16 hours. The solventwas concentrated under reduced pressure and the residue was diluted with10% aqueous citric acid and extracted with ethyl acetate. The organiclayer was washed with saturated brine and dried over anhydrous magnesiumsulfate. Unpurified title compound (330 mg) was obtained as yellow oilysubstance and used in the following reaction without being purified.

(B) (E)3-[2-{4-[2-(2-tert-Butoxycarbonylaminothiazol-5-yl)-3-pyridin4-yl-acryloyl]-piperazin-1-yl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl}-2-propenoicacid tert-butyl ester

A solution ofN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-3-{(E)-²-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-2-piperazino-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(297 mg) and (E)-2-(2-tert-butoxycarbonylaminothiazole-5-yl)-3-pyridin-4-yl-2-propenoic acid (180 mg) inN,N-dimethylformamide (2 ml) and methylene chloride (2 ml) was addedwith a solution of WSCI.HCl (150 mg, 0.780 mmol) in methylene chloride(6 ml) with ice cooling. The reaction mixture was stirred at roomtemperature for 20 hours, and then the solvent was concentrated underreduced pressure. The residue was purified by preparative TLC (4 plates,dichloromethane:methanol=9:1), preparative TLC (3 plates, ethyl acetate)and preparative TLC (1 plate, dichloromethane:methanol=9:1) to obtainthe title compound (27.1 mg) as orange foamy syrup.

¹H-NMR (CDCl₃) δ: 1.33 (9H, s), 1.49 (9H, s), 1.56 (9H, s), 2.96-3.10(1H, m), 3.24-4.01 (7H, m), 6.58 (1H, s), 6.94 (1H, s), 7.08 (1H, d,J=15.7Hz), 7.22-7.40 (5H, m), 7.49 (1H, d, J=7.3Hz), 7.85 (1H, s),8.60-8.82 (3H, m), 8.94 (1H, d, J=6.8Hz).

(C)(E)-3-[2-{4-[2-(2-Aminothiazol-5-yl)-3-pyridin-4-yl-acryloyl]piperazin-1-yl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(E)-3-[2-{4-[2-(2-tert-Butoxycarbonylaminothiazol-5-yl)-3-pyridin-4-yl-acryloyl]piperazin-1-yl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid tert-butyl ester (27.1 mg, 31.2 μmol) was dissolved in 4 N solutionof hydrochloric acid in dioxane (1 ml) and stirred at room temperaturefor 4 hours. The reaction mixture was subjected to azeotropy withtoluene and concentrated, and the residue was dissolved in methylenechloride (2 ml) again, added with trifluoroacetic acid (2 ml) andstirred at room temperature for 8 hours. The reaction mixture wassubjected to azeotropy with toluene and dried under reduced pressure,and the residue was added with ether. The precipitated solid wascollected by filtration and dried at 40° C. under reduced pressure toobtain the title compound (23.4 mg) as orange solid.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 3.27-4.00 (8H, m), 6.88 (1H, s), 6.95(1H, d, J=15.7Hz), 7.01 (1H, s), 7.34 (1H, s), 7.42 (1H, d, J=15.7Hz),7.66 (1H, d, J=7.6Hz), 7.87 (2H, d, J=6.4Hz), 8.17 (1H, s), 8.78 (2H, d,J=6.4Hz), 8.94 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 2968, 1678, 1633, 1593, 1520, 1439, 1365, 1284, 1232,1201, 1141, 1099, 1065, 1003.

ES-MS: m/z: 712 (MH⁺).

Anal. Calcd. for C₃₄H₃₃N₉O₅S₂.4HCl.2H₂O: C, 45.44; H, 4.77; N, 14.10.Found: C, 46.11; H, 4.85; N, 12.46.

Example 251(E)-3-[2-{4-[2-(2-Amino-1-methyl-1H-imidazol4-yl)-2-oxo-acetyl]-piperazin-1-yl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

(A)(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-{2-[1-methyl-2-(tritylamino)-1H-imidazol-4-yl]-2-oxo-acetyl}piperazin-1-yl)-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl]-2-propenoicacid tert-butyl ester

A solution ofN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-2-piperazino-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(456 mg) and [1-methyl-2-(tritylamino)-1H-imidazol-4-yl]-oxo-acetic acid(329 mg, 0.798 mmol) in N,N-dimethylformamide (2 ml) and methylenechloride (2 ml) was added with a solution of WSCI.HCl (230 mg, 1.20mmol) in methylene chloride (6 ml) with ice cooling and stirred at roomtemperature for 20 hours, and the solvent was concentrated under reducedpressure. The residue was purified by silica gel column chromatography(20 g, chloroform:methanol=99:1→98:2) to obtain the title compound (96.5mg) as orange foamy syrup.

¹H-NMR (CDCl₃) δ: 1.32 (9H, s), 1.51 (9H, s), 3.15 (5H, s), 3.27 (2H,s), 3.52 (2H, s), 3.65 (2H, s), 4.97 (1H, br), 6.60 (1H, s), 7.09-7.44(19Hm), 7.91 (1H, s), 8.91-8.95 (1H, m).

(B)(E)-3-[2-{4-[2-(2-Amino-1-methyl-1H-imidazol-4-yl)-2-oxo-acetyl]piperazin-1-yl}-8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid

A solution of(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-{2-[1-methyl-2-(tritylamino)-1H-imidazol-4-yl]-2-oxo-acetyl}piperazin-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid tert-butyl ester (96.5 mg) in dichloromethane (2 ml) was added withtrifluoroacetic acid (2 ml) with ice cooling and stirred at roomtemperature for 4 hours. The reaction mixture was concentrated underreduced pressure and subjected to azeotropy with 4 N solution ofhydrochloric acid in dioxane and toluene. The residue was added withether, and the precipitated solid was collected by filtration and driedat 40° C. under reduced pressure to obtain the title compound (70.4 mg)as orange solid.

¹H-NMR (DMSO-d₆) δ: 1.30 (9H, s), 3.15-3.80 (11H, m), 6.87 (1H, br),6.96 (1H, d, J=15.6Hz), 7.46 (1H, d, J=15.4Hz), 7.67 (1H, d, J=7.3Hz),8.10-8.28 (4H, m), 8.95 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 2966, 1678, 1639, 1593, 1516, 1440, 1363, 1282, 1234,1173, 1099, 1065, 1007.

ES-MS m/z: 634 (MH⁺).

m.p.: 210-216° C.

Anal. Calcd. for C₂₉H₃₁N₉O₆S.2HCl.1.5H₂O: C, 47.48; H, 4.95; N, 17.18.Found: C, 47.66; H, 5.15; N, 16.35.

Example 252 Carbamic acid1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-(2-dimethylaminoethylaminocarbonyl)vinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}-(R)-piperidin-3-yl-ester

(A) (R)-N-tert-Butoxycarbonyl-3-hydroxypiperidine

A solution of (R)-3-hydroxypiperidine (5.00 g) in 1,4-dioxane (100 ml)and saturated aqueous sodium hydrogencarbonate (100 ml) was added with(BOC)₂O (7.93 g) with ice cooling and stirred at room temperature for 15hours. The solvent was concentrated under reduced pressure, and theresidue was diluted with 10% aqueous citric acid and extracted withmethylene chloride. The organic layer was dried over anhydrous magnesiumsulfate, and the solvent was concentrated under reduced pressure toobtain unpurified title compound (7.58 g). The compound was used in thefollowing reaction without being further purified.

¹H-NMR (CDCl₃) δ: 1.46 (9H, s), 1.49-1.93 (5H, m), 3.04-3.17 (2H, m),3.51 (1H, br), 3.68-3.76 (2H, m).

(B) (R)-N-tert-Butoxycarbonyl-3-aminocarbonyloxypiperidine

A solution of (R)-N-tert-butoxycarbonyl-3-hydroxypiperidine (2.00 g) inethyl acetate (40 ml) was added with trichloroacetyl isocyanate (1.18ml) with ice cooling and stirred with ice cooling for 30 minutes. Thereaction mixture was further added with trichloroacetyl isocyanate(0.592 ml) with ice cooling and stirred with ice cooling for 30 minutes.The solvent was concentrated under reduced pressure, and a suspension ofthe residue in methanol (40 ml) and water (8 ml) was added with sodiumformate (2.03 g) and stirred at room temperature for 15 hours. Thereaction mixture was further added with sodium formate (2.03 g), stirredat room temperature for 4 hours, added with sodium formate (2.03 g),stirred at room temperature for 2 hours, added with sodium formate (2.03g), and stirred at room temperature for 18 hours. The solvent wasconcentrated under reduced pressure, and the residue was diluted withethyl acetate. The organic layer was washed with 1 N hydrochloric acid,saturated aqueous sodium hydrogencarbonate and saturated brine and driedover anhydrous magnesium sulfate. The solvent was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (100 g, hexane:ethyl acetate=2:1→1:1) to obtain the titlecompound (2.34 g)

¹H-NMR (CDCl₃) δ: 1.46 (9H, s), 1.47-1.92 (4H, m), 3.25-3.60 (4H, m),4.53-4.71 (3H, m).

(C) (R)-3-Aminocarbonyloxypiperidine hydrochloride

(R)-N-tert-Butoxycarbonyl-3-aminocarbonyloxypiperidine (2.34 g) wasdissolved in 4 N solution of hydrochloric acid in dioxane (10 ml) withice cooling and stirred at room temperature for 2 hours. The solvent wasremoved by azeotropy with toluene, and the residue was dried over underreduced pressure to obtain unpurified title compound (2.27 g). Thiscompound was used in the following reaction without being furtherpurified.

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.51-1.94 (4H, m), 2.81-3.27 (4H, m),4.69-4.78 (1H, m), 6.59 (2H, s), 9.11-9.58 (2H, m).

(D)(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-((R)-3-aminocarbonyloxypiperidin-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid tert-butyl ester

A solution of tert-Butyl(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoate(1.50 g) in N,N-dimethylformamide (100 ml) was added withdimethylaminopyridine (585 mg) and p-toluenesulfonyl chloride (913 mg)and stirred at room temperature for 2 hours. The reaction mixture wasfurther added with triethylamine (2.22 ml) and(R)-3-aminocarbonyloxypiperidine hydrochloride (2.27 g) and stirred atroom temperature for 22 hours. The solvent was concentrated underreduced pressure, and the residue was purified by silica gel columnchromatography (250 g,chloroform→chloroform:methanol=99:1→98:2→97:3→96:4, and 250 g,chloroform→chloroform:methanol=99:1→95:5) to obtain the title compound(1.69 g) as orange solid.

¹H-NMR (CDCl₃) δ: 1.29 (9H, d, J=3.9Hz), 1.46 (9H, d, J=3.7Hz), 1.66(2H, m), 1.83-2.05 (2H, m), 3.41-3.62 (2H, m), 3.84-3.95 (1H, m),4.49-4.66 (2H, m), 6.47 (2H, br), 6.83-6.95 (1H, m), 7.33-7.47 (1H, m),7.54-7.63 (1H, m), 7.94 (1H, s), 8.19 (1H, s), 8.82-8.91 (1H, m), 13.03(1H, br).

(E)(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-((R)-3-aminocarbonyloxypiperidin-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid hydrochloride

A solution of(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-((R)-3-aminocarbonyloxypiperidin-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid tert-butyl ester (1.69 g) in methylene chloride (10 ml) was addedwith TFA (10 ml) with ice cooling and stirred at room temperature for 2hours. The solvent was concentrated under reduced pressure and thereaction mixture was subjected to azeotropy with 4 N solution ofhydrochloric acid in dioxane. The residue was added with ether, and theprecipitated solid was collected by filtration, washed with ether anddried at 40° C. over under reduced pressure to obtain the title compound(1.26 g) as orange solid.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.58-2.05 (4H, m), 2.72-2.93 (1H, m),3.49-3.96 (3H, m), 4.52-4.68 (1H, m), 6.44 (2H, br), 6.87 (1H, s), 6.93(1H, d, J=15.4Hz), 7.44 (1H, s), 7.47 (1H, d, J=8.1Hz), 7.59-7.63 (2H,m), 8.19 (1H, s), 8.90 (1H, d, J=7.3Hz).

(F) Carbamic acid1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-[(E)-2-(2-dimethylaminoethylaminocarbonyl)vinyl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl}-(R)-piperidin-3-yl-ester

A solution of(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-((R)-3-aminocarbonyloxypiperidin-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid hydrochloride (100 mg) and N,N-dimethylethylenediamine (20.9 μl) inmethylene chloride (2 ml) and DMF (1 ml) was added withdiisopropylethylamine (75.5 ,μl), HOBt (25.8 mg) and WSCD.HCl (36.5 mg)with ice cooling and stirred at room temperature for 26 hours. Thesolvent was concentrated under reduced pressure, and the residue waspurified by preparative TLC (3 plates, lower layer ofchloroform:methanol:water=8:3:1) and preparative TLC (2 plates, lowerlayer of chloroform:methanol:water=8:3:1) to obtain the title compound(75.3 mg) as orange solid.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.61-1.76 (2H, m), 1.83-2.03 (2H, m),2.24 (6H, s), 2.39-2.48 (2H, m), 3.15-3.58 (6H, m), 3.83-3.91 (1H, m),4.57-4.67 (1H, m), 6.48 (1H, br), 6.85 (1H, s), 7.17 (1H, d, J=15.4Hz),7.22-7.30 (1H, m), 7.34 (1H, d, J=15.2Hz), 7.54 (0.5H, d, J=7.8Hz), 7.61(1H, dd, J=1.7, 7.3Hz), 7.79 (0.5H, d, J=7.8Hz), 8.06 (1H, t, J=5.6Hz),8.20 (1H, s), 8.89 (1H, d, J=7.3Hz).

ES-MS: m/z: 612 (MH⁺).

Anal. Calcd. for C₂₉H₃₈N₈O₅S.1.7H₂O: C, 54.31; H, 6.51; N, 17.47. Found:C, 54.12; H, 6.34; N, 17.86.

Example 2532-[4-(3-Aminopropionyl)-piperazin-1-yl]-4-oxo-3-[(E)-2-(1-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide

(A)3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-2-piperazin-1-yl-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide

tert-Butyl4-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-1-piperazinecarboxylate (100 mg) was dissolved in formic acid (2 ml) and stirred atroom temperature for 1 hour and 30 minutes. The solvent was concentratedunder reduced pressure by azeotropy with chloroform, and the residue wasdiluted with saturated aqueous sodium hydrogencarbonate and extractedwith chloroform. The organic layer was dried over anhydrous magnesiumsulfate, and the solvent was concentrated under reduced pressure toobtain unpurified title compound (76.6 mg). This compound was used inthe following reaction without being further purified.

¹H-NMR (DMSO-d₆) δ: 1.29 (9H, s), 2.91 (4H, m), 3.54 (4H, br), 5.63 (2H,s), 6.77 (1H, s), 6.94 (1H, d, J=8.5Hz), 7.26 (1H, d, J=8.8Hz), 7.64(1H, d, J=15.6Hz), 7.66-7.68 (1H, m), 7.76 (1H, d, J=16.6Hz), 8.16 (1H,m), 8.93 (1H, d, J=7.3Hz).

(B){3-[4-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperazin-1-yl]-3-oxopropyl}carbamicacid tert-butyl ester

A solution of3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-2-piperazin-1-yl-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide (76.6 mg) and Boc-β-alanine (25.4mg) in methylene chloride (2 ml) and N,N-dimethylformamide (2 ml) wasadded with WSCD.HCl (25.8 mg) with ice cooling and stirred at roomtemperature for 17 hours. The solvent was concentrated under reducedpressure, and the residue was diluted with chloroform and washed with10% aqueous citric acid, saturated aqueous sodium hydrogencarbonate andsaturated brine. The organic layer was dried over anhydrous magnesiumsulfate and concentrated under reduced pressure. The residue waspurified by preparative TLC (chloroform:methanol=40:1, developed twice)to obtain the title compound (64.7 mg).

¹H-NMR (CDCl₃) δ: 1.42 (9H, s), 1.44 (9H, s), 2.47-2.59 (2H, m),3.36-3.69 (10H, m), 3.80 (3H, s), 5.28 (1H, br), 5.57 (2H, s), 6.56 (1H,s), 6.94 (2H, d, J=7.3Hz), 7.40 (3H, d, J=7.1Hz), 7.78 (1H, d,J=15.4Hz), 7.89 (1H, s), 7.95 (1H, d, J=15.4Hz), 8.86 (1H, br).

(C)2-[4-(3-Aminopropionyl)piperazin-1-yl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide

{3-[4-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperazine-1-yl]-3-oxopropyl}carbamicacid tert-butyl ester (64.7 mg) was dissolved in 4 N solution ofhydrochloric acid in dioxane (5 ml) and stirred at room temperature for30 minutes. The solvent was concentrated under reduced pressure byazeotropy with chloroform to obtain unpurified title compound (69.6 mg).This compound was used in the following reaction without being furtherpurified.

(D)2-[4-(3-Aminopropionyl)-piperazin-1-yl]-4-oxo-3-[(E)-2-(1-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl) amide

2-[4-(3-Aminopropionyl)piperazin-1-yl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butyl-thiazol-2-yl)amide (69.6 mg) was dissolved intrifluoroacetic acid (2 ml) and stirred at 60° C. for four hours. Thesolvent was concentrated under reduced pressure by azeotropy withchloroform. The residue was added with methanol, and the insolublematters were removed by filtration. The residue was purified by HPLC toobtain the title compound (19.2 mg) as orange solid.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 2.72 (2H, t, J=5.9Hz), 3.04 (2H, t,J=5.9Hz), 3.50-3.77 (8H, m), 6.79 (1H, s), 7.28 (1H, d, J=15.9Hz), 7.68(1H, d, J=7.3Hz), 7.86 (1H, d, J=15.9Hz), 8.18 (1H, s), 8.97 (1H, d,J=7.3Hz).

IR (ATR) cm⁻¹: 2964, 2866, 1657, 1626, 1514, 1431, 1371, 1313, 1227,1144, 1103, 1068, 1018.

ES-MS: m/z: 578 (MH⁺).

Anal. Calcd. for C₂₆H₃₁N₁₁O₃S.3HCOOH-4H₂O: C, 44.21; H, 5.76; N, 19.56.Found: C, 43.87; H, 5.18; N, 20.01.

In a similar manner, the following compounds were synthesized.

Example 2542-[4-(4-Aminobutyryl)piperazin-1-yl]-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.73-1.87 (2H, m), 2.45-2.58 (2H, m),2.80-2.90 (2H, m), 3.49-3.75 (8H, m), 6.81 (1H, s), 7.28 (1H, d,J=15.9Hz), 7.67 (1H, d, J=7.3Hz), 7.86 (1H, d, J=15.9Hz), 8.19 (1H, s),8.97 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 2962, 1658, 1620, 1516, 1431, 1365, 1311, 1225, 1132,1103, 1066, 1016.

ES-MS: m/z: 592 (MH⁺).

Anal. Calcd. for C₂₇H₃₃N₁₁O₃S.3HCOOH-2.5H₂O: C, 46.50; H, 5.72; N,19.89. Found: C, 46.41; H, 5.69; N, 19.46.

Example 2552-[4-(5-Aminopentanoyl)piperazin-1-yl]-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.59 (4H, br), 2.41 (2H, br), 2.81(2H, br), 3.54 (4H, br), 3.67 (4H, br), 6.78 (1H, s), 7.27 (1H, d,J=16.3Hz), 7.68 (1H, d, J=7.6Hz), 7.86 (1H, d, J=16.3Hz), 8.18 (1H, s),8.97 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 2964, 1658, 1618, 1516, 1429, 1365, 1311, 1254, 1225,1206, 1132, 1103, 1066, 1016.

ES-MS: m/z: 606 (MH⁺).

Anal. Calcd. for C₂₈H₃₅N₁₁O₃S.3HCOOH.4H₂O: C, 45.63; H, 6.05; N, 18.89.Found: C, 45.86; H, 5.95; N, 18.32.

Example 2562-[4-(6-Aminohexanoyl)-piperazin-1-yl]-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.25-1.40 (m, 2H), 1.31 (9H, s), 1.50-1.61(4H, m), 2.31-2.42 (2H, m), 2.76-2.85 (2H, m), 3.54 (4H, br), 3.66 (4H,br), 6.79 (1H, s), 7.27 (1H, d, J=16.2Hz), 7.67 (1H, dd, J=1.7, 7.3Hz),7.85 (1H, d, J=16.2Hz), 8.19 (1H, s), 8.97 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 2958, 2864, 1658, 1618, 1516, 1427, 1365, 1311, 1250,1225, 1134, 1103, 1068, 1018.

ES-MS: m/z: 620 (MH⁺).

Anal. Calcd. for C₂₉H₃₇N₁₁O₃S.3HCOOH.2.5H₂O: C, 47.86; H, 6.03; N,19.19. Found: C, 47.76; H, 6.05; N, 19.12.

Example 2572-[4-(2-Aminoacetyl)piperazin-1-yl]-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.31 (9H, s), 3.16-3.77 (8H, m), 3.93 (2H,s), 6.85 (1H, s), 7.32 (1H, d, J=16.0Hz), 7.68 (1H, d, J=7.3Hz), 7.89(1H, d, J=16.0Hz), 8.20 (1H, s), 8.98 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 2960, 2359, 1658, 1514, 1431, 1371, 1309, 1244, 1203,1134, 1103, 1065, 1013.

ES-MS: m/z: 564 (MH⁺).

Anal. Calcd. for C₂₅H₂₉N₁₁O₃S.3HCOOH.3H₂O: C, 44.50; H, 5.47; N, 20.39.Found: C, 44.99; H, 5.47; N, 20.75.

Example 2582-{4-[2-(2-Aminoacetylamino)acetyl]piperazin-1-yl}-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 3.07-3.72 (10H, m), 4.12 (2H, s), 6.86(1H, s), 7.34 (1H, d, J=16.0Hz), 7.67 (1H, d, J=6.9Hz), 7.87 (1H, d,J=16.0Hz), 8.22 (1H, s), 8.50 (1H, br), 8.99 (1H, d, J=7.6Hz).

IR (ATR) cm⁻¹: 2962, 2866, 1657, 1637, 1516, 1431, 1365, 1227, 1205,1103, 1066, 1016.

ES-MS: m/z: 621 (MH⁺).

Anal. Calcd. for C₂₇H₃₂N₁₂O₄S.3HCOOH.2H₂O: C, 45.34; H, 5.33; N, 21.15.Found: C, 45.49; H, 5.51; N, 21.32.

Example 259{[2-(4-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperazin-1-yl)-2-oxoethylaminocarbonyl]methyl}trimethylammonium

(A) (2-Dimethylaminoacetylamino)acetic acid tert-butyl ester

A solution of glycine tert-butyl ester hydrochloride (1.63 g) andsarcosine (1.00 g) in methylene chloride (20 ml) andN,N-dimethylformamide (20 ml) was added with triethylamine (2.03 ml),HOBt (1.44 g) and WSCD.HCl (2.04 g) with ice cooling and stirred at roomtemperature for 14 hours. The solvent was concentrated under reducedpressure, and the residue was diluted with ethyl acetate and washed with10% aqueous citric acid, saturated aqueous sodium hydrogencarbonate andsaturated brine. The organic layer was dried over anhydrous magnesiumsulfate and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography(chloroform→chloroform:methanol=95:5) to obtain the title compound (2.08g) as pale yellow oily substance.

¹H-NMR (CDCl₃) δ: 1.48 (9H, s), 2.33 (6H, s), 2.99 (2H, s), 3.97 (2H, d,J=5.6Hz), 7.57 (1H, br).

(B) (2- Dimethylaminoacetylamino)acetic acid

(2-Dimethylaminoacetylamino)acetic acid tert-butyl ester (2.08 g) wasdissolved in trifluoroacetic acid (10 ml) and stirred at roomtemperature for 4 hours. The solvent was concentrated under reducedpressure by azeotropy with chloroform and ethanol to obtain unpurifiedtitle compound (2.93 g). This compound was used in the followingreaction without being further purified.

¹H-NMR (CDCl₃) δ: 2.94 (6H, d, J=2.2Hz), 4.00 (4H, d, J=2.0Hz).

(C)2-{4-[2-(2-Dimethylaminoacetylamino)acetyl]piperazin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylic acid (4-tert-butylthiazol-2-yl)amide

A solution ofN⁸-[4-(tert-butyl)-1,3-thiazol-2-yl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-2-piperazino-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(92.9 mg) and (2-dimethylaminoacetylamino)acetic acid (41.5 mg) inmethylene chloride (4 ml) and N,N-dimethylformamide (1 ml) was addedwith triethylamine (48.0 μl), HOBt (20.5 mg) and WSCD.HCl (29.0 mg) withice cooling and stirred at room temperature for 19 hours. The solventwas concentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (10 g,chloroform→chloroform:methanol=99:1→98:2→95:5→90:10) and preparative TLC(chloroform:methanol=10:1) to obtain the title compound (76.7 mg) asorange foamy syrup.

¹H-NMR (CDCl₃) δ: 1.39 (9H, s), 2.30 (6H, s), 3.00 (2H, s), 3.50-3.81(8H, m), 3.80 (3H, d, J=0.5Hz), 4.16 (2H, d, J=4.6Hz), 5.56 (2H, s),6.58 (1H, s), 6.92 (2H, d, J=8.7Hz), 7.38 (2H, d, J=8.7Hz), 7.49 (1H, d,J=7.6Hz), 7.77 (1H, d, J=15.5Hz), 7.96 (1H, d, J=15.5Hz), 7.95-8.03 (2H,m), 8.94 (1H, d, J=7.3Hz).

(D)2-{4-[2-(2-Dimethylaminoacetylamino)acetyl]piperazin-1-yl}-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxylic acid (4-tert-butylthiazol-2-yl)amide

2-{4-[2-(2-Dimethylaminoacetylamino)acetyl]piperazin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide (76.7=mg) was dissolved intrifluoroacetic acid (2 ml) and stirred at room temperature for 4 hours.The solvent was concentrated under reduced pressure by azeotropy withethanol and formic acid. The residue was added with methanol, and theinsoluble matters were removed by filtration to obtain the unpurifiedcompound (80.9 mg). This compound (70.9 mg) was purified by HPLC toobtain the title compound (22.8 mg) as orange solid.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 2.77 (6H, s), 3.56-3.81 (10H, m), 4.20(2H, s), 6.72 (1H, s), 7.47 (1H, d, J=16.4Hz), 7.61 (1H, d, J=7.1Hz),7.95 (1H, d, J=16.4Hz), 7.97-8.04 (2H, m), 8.97 (1H, d, J=7.6Hz).

(E){[2-(4-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperazin-1-yl)-2-oxoethylaminocarbonyl]methyl}trimethylammonium

A solution of2-{4-[2-(2-dimethylaminoacetylamino)acetyl]piperazin-1-yl}-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide (22.8 mg) in N,N-dimethylformamide(1 ml) was added with methyl iodide (100 a I) and left stand at 5° C.for 21 hours. The reaction mixture was further added with methyl iodide(100 μl), left stand at 5° C. for 16 hours, and then the solvent wasconcentrated under reduced pressure by azeotropy with ethanol. Theresidue was added with ether, and the precipitated solid was collectedby filtration, washed with ether and dried at 40° C. under reducedpressure to obtain the title compound (19.9 mg) as orange solid.

¹H-NMR (400 MHz, DMSO-d₆.CD₃OD) δ: 1.33 (9H, s), 2.56 (9H, s), 3.60-3.76(8H, m), 4.15 (4H, s), 6.86 (1H, br), 7.56 (1H, d, J=16.0Hz), 7.71 (1H,d, J=8.3Hz), 7.87 (1H, d, J=16.0Hz), 8.25 (1H, br), 8.74 (1H, br), 9.00(1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 2962, 2866, 2754, 1655, 1637, 1541, 1512, 1437, 1363,1333, 1281, 1227, 1165, 1101, 1051, 1016.

ES-MS: m/z: 663 (MH⁺).

In a similar manner, the following compound was synthesized.

Example 260[2-(4-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperazin-1-yl)-2-oxoethyl]trimethylammonium

¹H-NMR (CDCl₃) δ: 1.33 (9H, s), 3.38 (9H, s), 3.54 (6H, br), 3.69 (2H,br), 4.50 (2H, s), 6.66 (1H, s), 7.36 (1H, d, J=15.9Hz), 7.54 (1H, br),7.80-7.94 (2H, m), 8.85 (1H, br).

Anal. Calcd. for C₂₈H₃₆N₁₁O₃SI.4.5TFA.1.5H₂O: C, 34.86; H, 3.44; N,12.09; S, 2.52; F, 20.12. Found: C, 35.25; H, 3.11; N, 12.04; S, 2.34;F, 19.76.

IR (ATR) cm⁻¹: 3415, 2972, 1672, 1541, 1512, 1439, 1367, 1254, 1203,1176, 1124.

ES-MS: m/z: 606 (M⁺).

Example 2612-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetylamino]ethyl}trimethylammonium

(A)[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]aceticacid

A solution of[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]aceticacid ethyl ester (164 mg) in THF (3 ml) was added with 1 N aqueoussodium hydroxide (346 μl) and stirred at room temperature for 1 hour andat 50° C. for 1 hour. The reaction mixture was further added with 1 Naqueous sodium hydroxide (346 μl), stirred at 50° C. for 16 hours, addedwith 1 N aqueous sodium hydroxide (115 μl) and stirred at 50° C. for 1hour. The reaction mixture was neutralized by adding 1 N hydrochloricacid, diluted with saturated brine and extracted with chloroform. Theorganic layers were combined and dried over anhydrous sodium sulfate toobtain the title compound (130 mg, 82%). This compound was used in thefollowing reaction without being purified.

¹H-NMR (CDCl₃—CD₃OD) δ: 1.36 (9H, s), 1.30-1.41 (1H, m), 1.63-1.81 (2H,m), 1.93-2.03 (1H, m), 2.15-2.33 (3H, m), 2.96-3.06 (1H, m), 3.16-3.27(1H, m), 3.78 (3H, m), 4.00 (1H, d, J=11.7Hz), 4.23 (1H, d, J=12.7Hz),5.60 (2H, s), 6.67 (1H, s), 6.92 (2H, d, J=8.6Hz), 7.35 (2H, d,J=8.6Hz), 7.58 (1H, dd, J=1.8, 7.3Hz), 7.65 (1H, d, J=15.6Hz), 7.80 (1H,d, J=15.6Hz), 8.03 (1H, d, J=1.2Hz), 8.93 (1H, d, J=7.3Hz).

(B)2-{3-[(2-Dimethylaminoethylaminocarbonyl)methyl]piperidin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide

A solution of[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]aceticacid (130 mg) and N,N-dimethylethylenediamine (20.8 μg) in methylenechloride (2 ml) and N,N-dimethylformamide (2 ml) was added withtriethylamine (39.7 μl), HOBt (28.2 mg) and WSCD.HCl (40.0 mg) with icecooling and stirred at room temperature for 16 hours. The solvent wasconcentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (10 g,chloroform→chloroform:methanol=98:2→96:4→94:6→98:2→90:10) andpreparative TLC (3 plates, chloroform:methanol=10:1, developed twice) toobtain the title compound (129 mg, 90%) as orange foamy syrup.

¹H-NMR (CDCl₃) δ: 1.27-1.41 (1H, m), 1.35 (9H, s), 1.49-1.77 (2H, m),1.88-1.99 (1H, m), 2.12-2.32 (3H, m), 2.26 (6H, s), 2.50-2.61 (2H, m),2.86-2.99 (1H, m), 3.11-3.23 (1H, m), 3.39-3.44 (2H, m), 3.77 (3H, s),3.90-4.11 (1H, m), 4.09-4.20 (1H, m), 5.51 (2H, s), 6.56 (1H, s), 6.88(2H, d, J=8.7Hz), 6.93 (1H, br), 7.35 (2H, d, J=8.7Hz), 7.49 (1H, dd,J=1.7, 7.3Hz), 7.66 (1H, d, J=15.4Hz), 7.83 (1H,d, J=15,4Hz), 7.90 (1H,s), 8.90 (1H, d, J=7.6Hz).

(C)(2-{2-[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]acetylamino}ethyl)trimethylammonium

A solution of2-{3-[(2-dimethylaminoethylaminocarbonyl)methyl]piperidin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide (129 mg) in N,N-dimethylformamide(4 ml) was added with methyl iodide (1 ml) and left stand at 5° C. for16 hours. The solvent was evaporated by azeotropy with toluene, and theresidue was purified by preparative TLC (2 plates,chloroform:methanol:water=8:3:0.5, developed twice) to obtain the titlecompound (132 mg, 86%) as orange solid.

¹H-NMR (CD₃OD-CDCl₃) δ: 1.23-1.41 (1H, m), 1.34 (9H, s), 1.60-1.84 (2H,m), 1.89-2.00 (1H, m), 2.10-2.28(3H, m), 2.85-2.99 (1H, m), 3.20 (9H,s), 3.25-3.33 (1H, m), 3.40-3.52 (2H, m), 3.55-3.67 (2H, m), 3.75 (3H,s), 3.95-4.10 (2H, m), 5.58 (2H, s), 6.71 (1H, s), 6.92 (2H, d,J=8.8Hz), 7.33 (2H, d, J=8.8Hz), 7.54 (1H, dd, J=1.7, 7.3Hz), 7.59 (1H,d, J=15.6Hz), 7.72 (1H, d, J=15.6Hz), 7.83-7.92 (1H, m), 8.88 (1H, d,J=7.3Hz).

(D)2-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetylamino]ethyl}trimethylammonium

A solution of(2-{2-[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]acetylamino}ethyl)trimethylammonium(132 mg) in trifluoroacetic acid (3 ml) and stirred at room temperaturefor 1 hour and at 80° C. for 5 hours. The solvent was evaporated byazeotropy with toluene, and residue was purified by preparative TLC (2plates, chloroform:methanol:water=8:3:0.5) to obtain the title compound(73.7 mg) as red solid.

¹H-NMR (400 MHz, DMSO-d₆—CD₃OD) δ: 1.25-1.40 (1H, m), 1.35 (9H, s),1.70-1.98 (3H, m), 2.03-2.28 (3H, m), 2.90-2.99 (1H, m), 3.12 (9H, s),3.08-3.57 (5H, m), 3.92-4.01 (1H, m), 4.15-4.24 (1H, m), 6.73 (1H, s),7.36 (1H, d, J=16.1Hz), 7.55(1H, dd, J=2.0, 7.6Hz), 7.93 (1H, d,J=16.1Hz), 7.96-7.99 (1H, m), 8.94 (1H, d, J=7.6Hz).

IR (ATR) cm⁻¹: 3037, 2956, 2929, 2858, 1655, 1514, 1421, 1375, 1309,1254, 1201, 1173, 1124, 1103.

ES-MS: m/z: 648 (M⁺).

Anal. Calcd. for C₃₁H₄₂N₁₁O₃S.0.5.I0.5TFA.0.75H₂O: C, 49.07; H, 5.66; N,16.97; S, 4.09; F, 3.64. Found: C, 49.44; H, 5.99; N, 19.25; S, 4.18; F,3.21.

m.p.: 201-205° C.

Example 262{2-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetylamino]ethyl}aminocarbonylmethyldimethylammonium

(A)(2-{2-[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]acetylamino}ethyl)aminocarbonylmethyldimethylammonium

A solution of2-{3-[(2-dimethylaminoethylaminocarbonyl)methyl]piperidin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide (123 mg) in N,N-dimethylformamide(2 ml) was added with iodoacetamide (33.3 mg) and left stand at 5° C.for 16 hours. The reaction mixture was further added with iodoacetamide(27.2 mg), stirred at room temperature for 24 hours. The solvent wasevaporated by azeotropy with toluene, and then the residue was purifiedby preparative TLC (3 plates, chloroform:methanol:water=8:3:0.5) toobtain the title compound (158 mg, quantitative) as orange solid.

¹H-NMR (CD₃OD): δ 1.27-1.40 (1H, m), 1.34 (9H, s), 1.56-1.83 (2H, m),1.90-1.99 (1H, m), 2.12-2.27 (3H, m), 2.87-2.99 (1H, m), 3.08-3.19 (1H,m), 3.36 (6H, s), 3.57-3.80 (4H, m), 3.76 (3H, s), 3.88-4.08 (2H, m),4.21 (2H, s), 5.59 (2H, s), 6.71 (1H, s), 6.92 (2H, d, J=8.9Hz), 7.33(2H, d, J=8.9Hz), 7.54 (1H, dd, J=1.8, 7.6Hz), 7.58 (1H, d, J=15.5Hz),7.73 (1H, d, J=15.5Hz), 7.85-7.95 (1H, m), 8.89 (1H, d, J=7.6Hz).

(B){2-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetylamino]ethyl}aminocarbonylmethyldimethylammonium

(2-{2-[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-alpyrimidin-2-yl)piperidin-3-yl]acetylamino}ethyl)aminocarbonylmethyldimethylammonium(158 mg) was dissolved in trifluoroacetic acid (2 ml) and stirred at 80°C. for 5 hours. The solvent was evaporated by azeotropy with toluene,and the residue was purified by preparative TLC (2 plates,chloroform:methanol:water=8:3:0.5) to obtain the title compound (116 mg)as red solid.

¹H-NMR (400 MHz, CD₃OD.CDCl₃) δ: 1.28-1.41 (1H, m), 1.35 (9H, s),1.70-1.99 (3H, m), 2.01-2.29 (3H, m), 2.86-2.97 (1H, m), 3.09-3.17 (1H,m), 3.20-3.78 (4H, m), 3.97-4.06 (1H, m), 4.11 (2H, s), 4.14-4.23 (1H,m), 6.73 (1H, s), 7.36 (1H, d, J=16.4Hz), 7.56 (1H, d, J=7.6Hz), 7.91(1H, d, J=16.4Hz), 7.99 (1H, s), 8.95 (1H, d, J=7.6Hz).

IR (ATR) cm⁻¹: 3319, 3195, 2964, 1671, 1518, 1425, 1363, 1311, 1257,1201, 1176, 1126.

ES-MS: m/z: 691 (M⁺).

Anal. Calcd. for C₃₂H₄₃N₁₂O₄S.0.5I.2TFA.2.5H₂O: C, 42.03; H, 4.90; N,16.34; S, 3.12; F, 11.10. Found: C, 41.96; H, 4.51; N, 15.76; S, 3.04;F, 11.90.

m.p.: 175-185° C.

Example 263(2-{[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetyl]methylamino}ethyl)trimethylammonium

(A)2-(3-{[(2-Dimethylaminoethyl)methylaminocarbonyl]methyl}piperidin-1-yl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide

A solution of[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]aceticacid (140 mg) and N,N,N′-trimethylethylenediamine (26.7 μg) in methylenechloride (2 ml) and N,N-dimethylformamide (2 ml) was added withtriethylamine (42.9 μl), HOBt (30.5 mg) and WSCD.HCl (43.2 mg) with icecooling and stirred at room temperature for 14 hours. The solvent wasconcentrated under reduced pressure, and the residue was purified bypreparative TLC (3 plates, chloroform:methanol=10:1) to obtain the titlecompound (145 mg, 92%) as orange solid.

¹H-NMR (CDCl₃) δ: 1.28-1.42 (1H, m), 1.34 (9H, s), 1.57-1.79 (2H, m),1.90-2.00 (1H, m), 2.20-2.60 (4H, m), 2.25 (3H, s), 2.29 (3H, s), 2.92(1H, s), 2.95-3.10 (1H, m), 3.06 (3H, s), 3.18-3.31 (1H, m), 3.40-3.63(2H, m), 3.77 (3H, s), 3.90-4.00 (1H, m), 4.09-4.26 (1H, m), -5.51 (2H,s), 6.53-6.60 (1H,-m),:6.89 (2H, d, J=8.5Hz), 7.35 (2H, d, J=8.5Hz),7.45-7.54 (1H, m), 7.35 (1H, d, J=8.5Hz), 7.71 (1H, d, J=8.5Hz),7.90-7.96 (1H, m), 8.88-8.98 (1H, m).

(B)[2-({2-[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]acetyl}methylamino)ethyl]trimethylammonium

A solution of2-(3-{[(2-dimethylaminoethyl)methylaminocarbonyl]methyl}piperidin-1-yl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide (145 mg) in N,N-dimethylformamide(2 ml) was added with methyliodide (1 ml) and left stand at 5° C. for 12hours. The solvent was evaporated by azeotropy with toluene, and theresidue was purified by preparative TLC (2 plates,chloroform:methanol:water=8:3:0.5) to obtain the title compound (159 mg,92%) as red solid.

¹H-NMR (CD₃OD) δ: 1.25-1.41 (1H, m), 1.35 (9H, s), 1.61-1.84 (2H, m),1.90-2.00 (1H, m), 2.18-2.46 (3H, m), 2.98-3.03 (1H, m), 3.09 (3H, s),3.11-3.22 (1H, m), 3.19 (9H, s), 3.45-3.55 (2H, m), 3.70-3.80 (2H, m),3.76 (3H, s), 3.96-4.08 (2H, m), 5.60 (2H, s), 6.72 (1H, s), 6.92 (2H,d, J=8.8Hz), 7.33 (2H, d, J=8.8Hz), 7.56 (1H, dd, J=1.7, 7.3Hz), 7.62(1H, d, J=15.6Hz), 7.75 (1H, d, J=15.6Hz), 7.84-7.93 (1H, m), 8.90 (1H,d, J=7.6Hz).

(C)(2-{[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetyl]methylamino}ethyl)trimethylammonium

A solution of[2-({2-[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]acetyl}methylamino)ethyl]trimethylammonium(159 mg) in trifluoroacetic acid (2 ml) was stirred at room temperaturefor 1 hour and at 80° C. for 4 hours. The solvent was evaporated byazeotropy with toluene, and the residue was purified by preparative TLC(2 plates, chloroform:methanol:water=8:3:0.5) to obtain the titlecompound (139 mg) as red solid.

¹H-NMR (CD₃OD-CDCl₃) δ: 1.27-1.38 (1H, m), 1.34 (9H, s), 1.69-1.86 (2H,m), 1.90-1.99 (1H, m), 2.13-2.25 (2H, m), 2.33-2.42 (1H, m), 2.79-2.89(1H, m), 2.99 (3H, s), 3.00-3.38 (5H, m), 3.11 (9H, s), 3.94-4.03 (1H,m), 4.12-4.21 (1H, m), 6.72 (1H, s), 7.35 (1H, d, J=16.4Hz), 7.54 (1H,dd; J=1.7, 7.3Hz), 7.88-7.97 (1H, m), 7.92 (1H, d, J=16.4Hz), 8.92 (1H,d, J=7.3Hz).

IR (ATR) cm⁻¹: 2962, 2937, 2864, 1676, 1635, 1514, 1423, 1379, 1309,1255, 1203, 1174, 1124.

ES-MS: m/z: 662 (M⁺).

Anal. Calcd. for C₃₂H₄₄N₁₁O₃S.0.5I.2.5TFA 1.5H₂O: C, 42.78; H, 4.80; N,14.83; S, 3.09; F, 13.72. Found: C, 42.80; H, 4.56; N, 14.57; S, 3.11;F, 13.01.

m.p.: 163-168° C.

Example 264{3-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetylamino]propyl}aminocarbonylmethyldimethylammonium

(A)2-{3-[(3-Dimethylaminopropylaminocarbonyl)methyl]piperidin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylic acid (4-tert-butylthiazol-2-yl)amide

A solution of[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]aceticacid (250 mg) and N,N-dimethyl-1,3-propanediamine (46.0 μl) in methylenechloride (2 ml) and N,N-dimethylformamide (2 ml) was added withtriethylamine (76.5 g l), HOBt (54.3 mg) and WSCD.HCl (77.1 mg) with icecooling and stirred at room temperature for 16 hours. The solvent wasconcentrated under reduced pressure, and the residue was purified bysilica gel column chromatography (25 g,chloroform→chloroform:methanol=15:1→10:1→4:1) and preparative TLC (3plates, chloroform:methanol=4:1) to obtain the title compound (283 mg,90%) as orange foamy syrup.

¹H-NMR (CD₃OD-CDCl₃) δ: 1.28-1.41 (1H, m), 1.35 (9H, s), 1.65-2.00 (5H,m), 2.14-2.22 (3H, m), 2.68 (6H, s), 2.83-2.89 (2H, m), 2.92-2.99 (1H,m), 3.12-3.20 (3H, m), 3.76 (3H, s), 4.01-4.10 (2H, m), 5.61 (s, 2H),6.73 (1H, s), 6.93 (2H, d, J=8.8Hz), 7.35 (2H, d, J=8.8Hz), 7.58 (1H,dd, J=1.2, 7.3Hz), 7.64 (1H, d, J=15.6Hz), 7.79 (1H, d, J=15.6Hz),7.87-7.90 (1H, m), 8.94 (1H, d, J=7.3Hz).

(B)(3-{2-[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]acetylamino}propyl)aminocarbonylmethyldimethylammonium

A solution of2-{3-[(3-dimethylaminopropylaminocarbonyl)methyl]piperidin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide (142 mg) in N,N-dimethylformamide(3 ml) was added with iodoacetamide (37.2 mg) and stirred at roomtemperature for 12 hours. The reaction mixture was added withiodoacetamide (30.4 mg) and stirred at room temperature for 12 hours and30 minutes. The reaction mixture was further added with iodoacetamide(34.1 mg), stirred at room temperature for 13 hours and the solvent wasevaporated by azeotropy with toluene. The residue was diluted withsaturated aqueous sodium hydrogencarbonate and extracted withchloroform/methanol, and the organic layer was dried over anhydrousmagnesium sulfate. The solvent was concentrated under reduced pressure,and the residue was dissolved in N,N-dimethylformamide (3 ml), addedwith iodoacetamide (34.1 mg) and stirred at room temperature for 1 hour.The solvent was evaporated by azeotropy with toluene, and the residuewas purified by preparative TLC (2 plates,chloroform:methanol:water=8:3:0.5) to obtain the title compound (113 mg,64%) as orange solid.

¹H-NMR (CD₃OD.CDCl₃) δ: 1.26-1.41 (1H, m), 1.34 (9H, s), 1.65-1.84 (2H,m), 1.91-2.04 (3H, m), 2.12-2.28 (3H, m), 2.88-2.97 (1H, m), 3.09-3.24(3H, m), 3.29 (6H, s), 3.59-3.67 (2H, m), 3.76 (3H, s), 3.99-4.09 (2H,m), 4.12 (2H, s), 5.59 (2H, s), 6.71 (1H, s), 6.92 (2H, d, J=8.6Hz),7.33 (2H, d, J=8.6Hz), 7.52-7.57 (1H, m), 7.61 (1H, d, J=15.4Hz), 7.73(1H, d, J=15.4Hz), 7.91-7.94 (1H, m), 8.89 (1H, d, J=7.3Hz).

(C){3-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetylamino]propyl}aminocarbonylmethyldimethylammonium

A solution of(3-{2-[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]acetylamino}propyl)aminocarbonylmethyldimethylammonium(113 mg) in trifluoroacetic acid (2 ml) was stirred at 80° C. for 4hours and 30 minutes. The solvent was evaporated by azeotropy withtoluene, and the residue was purified by preparative TLC (2 plates,chloroform:methanol:water=8:3:0.5). The resulting fraction was subjectedto azeotropy with formic acid, and the residue was purified by HPLC toobtain the title compound (33.2 mg) as orange solid.

¹H-NMR (400 MHz, CD₃OD.CDCl₃) δ: 1.27-1.41 (1H, m), 1.35 (9H, s),1.67-1.99 (5H, m), 2.05-2.25 (3H, m), 2.90-3.21 (4H, m), 3.28 (6H, s),3.47-3.56 (2H, m), 3.92-4.02 (1H, m), 4.03-4.18 (1H, m), 4.09 (2H, s),6.72 (1H, s), 7.39 (1H, d, J=15.8Hz), 7.51-7.58 (1H, m), 7.84-7.95 (1H,m), 7.95 (1H, d, J=15.8Hz), 8.90 (1H, d, J=7.3Hz).

IR (ATR) cm⁻¹: 3049, 2960, 2929, 2854, 1653, 1514, 1421, 1375, 1308,1252, 1227, 1203, 1155, 1101, 1066.

ES-MS: m/z: 705 (M⁺)

Anal. Calcd. for C₃₃H₄₄N₁₂O₄S.HCOOH.1.5H₂O: C, 52.50; H, 6.35; N, 21.61.Found: C, 52.38; H, 6.50; N, 21.51.

m.p.: 186-193° C.

Example 265(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3-[(2-hydroxyethylaminocarbonyl)methyl]piperidin-1-yl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(A) 3-[(2-Hydroxyethylaminocarbonyl)methyl]piperidine-1-carboxylic acidtert-butyl ester

A solution of 3-carboxymethylpiperidine-1-carboxylic acid tert-butylester (583 mg) and ethanolamine (159 μl) in dichloromethane (5 ml) wasadded with triethylamine (502 μl), HOBt (389 mg) and WSCD.HCl (552 mg)at 0° C. Since insoluble matters were precipitated, the reaction mixturewas further added with N,N-dimethylformamide (5 ml) and stirred at roomtemperature for 12 hours. The reaction mixture was concentrated underreduced pressure by azeotropy with toluene, and the residue was dilutedwith ethyl acetate and washed with 10% aqueous citric acid, saturatedaqueous sodium hydrogencarbonate and saturated brine. The organic layerwas dried over anhydrous magnesium sulfate and concentrated underreduced pressure. The residue was purified by silica gel columnchromatography (50 g, chloroform:ethylacetate=1:1→chloroform:methanol=10:1) to obtain the title compound (643mg, 94%) as colorless oily substance.

¹H-NMR (CD₃OD) δ: 1.05-1.48 (1H, m), 1.44 (9H, s), 1.53-2.18 (6H, m),2.43-2.93 (2H, m), 3.13-3.35 (2H, m), 3.43-3.62 (2H, m), 3.68-3.94 (2H,m).

(B) N-(2-Hydroxyethyl)-2-piperidin-3-yl-acetamide hydrochloride

3-[(2-Hydroxyethylaminocarbonyl)methyl]piperidine-1-carboxylic acidtert-butyl ester (643 mg) was dissolved in 4 N hydrochloric acid indioxane (5 ml) and stirred at room temperature for 1 hour and 30minutes. The reaction mixture was subjected concentrated under reducedpressure by azeotropy with toluene to obtain the title compound (471 mg,94%) as colorless oily substance.

¹H-NMR (400 MHz, CDCl₃) δ: 1.22-1.38 (1H, m), 1.66-1.81 (1H, m),1.84-1.99 (2H, m), 2.12-2.27 (3H, m), 2.67-2.78 (1H, m), 2.85-2.97 (1H,m), 3.26-3.40 (4H, m), 3.59 (2H, t, J=5.7Hz).

(C)(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3-[(2-hydroxyethylaminocarbonyl)methyl]piperidin-1-yl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid tert-butyl ester

The following reaction was performed under argon atmosphere. A solutionof(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid tert-butyl ester (250 mg) in N,N-dimethylformamide (2 ml) was addedwith diphenylphosphoryl chloride (220 μl) and diisopropylethylamine (370μl) at 0° C. and stirred at 0° C. for 30 minutes. The reaction mixturewas added with N-(2-hydroxyethyl)-2-piperidin-3-yl-acetamidehydrochloride (237 mg) and diisopropylethylamine (370 μl) at the sametemperature and stirred at 80° C. for 2 hours. The reaction mixture wasreturned to room temperature, then diluted with water and extracted withchloroform. The organic layer was dried over anhydrous magnesiumsulfate, and the solvent was concentrated under reduced pressure byazeotropy with toluene. The residue was purified by silica gel columnchromatography (50 g, ethyl acetate) and preparative TLC (3 plates,ethyl acetate:methanol=99:1, developed twice) to obtain the titlecompound (106 mg, 31%) as orange foamy syrup.

¹H-NMR (CDCl₃) δ: 1.26-1.57 (1H, m), 1.35 (9H, s), 1.53 (9H, s),1.67-1.86 (2H, m), 1.91-2.02 (1H, m), 2.13-2.27 (3H, m), 2.93-3.04 (1H,m), 3.17-3.37 (3H, m), 3.62 (2H, t, J=5.9Hz), 3.93-4.02 (1H, m),4.12-4.20 (1H, m), 6.74 (1H, s), 6.94 (1H, d, J=15.6Hz), 7.47 (1H, d,J=15.6Hz), 7.55 (1H, dd, J=1.7, 7.3Hz), 7.95-8.00 (1H, m), 8.91 (1H, d,J=7.3Hz).

(D)(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3-[(2-hydroxyethylaminocarbonyl)methyl]piperidin-1-yl]-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{3-[(2-hydroxyethylaminocarbonyl)methyl]piperidin-1-yl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid tert-butyl ester (106 mg) was dissolved in 4 N hydrochloric acid indioxane (2 ml) and stirred at room temperature for 4 hours 30 minutes.Since solid was precipitated, the reaction mixture was added withmethanol. The solvent was concentrated under reduced pressure, and theresidue was purified by preparative TLC (2 plates,chloroform:methanol=10:1). Since a part of the product became methylester, this fraction was dissolved in tetrahydrofuran (3 ml), methanol(1 ml) and water (1 ml), added with lithium hydroxide monohydrate (10mg) and stirred at room temperature for 1 hour and at 50° C. for 1 hourand 30 minutes. The reaction mixture was further added with lithiumhydroxide monohydrate (total amount: 20 mg) with stirring at 50° C. for4 hours and 30 minutes and at 80° C. for 30 minutes. The reactionmixture was adjusted to pH 3 by adding 1 N hydrochloric acid, and thesolvent was concentrated under reduced pressure. The residue and theaforementioned fraction were purified by preparative TLC (3 plates,chloroform:methanol=4:1) to obtain the title compound (39.7 mg) asyellow solid.

¹H-NMR (CD₃OD-CDCl₃) δ: 1.26-1.44 (1H, m), 1.35 (9H, s), 1.66-1.85 (2H,m), 1.91-1.99 (1H, m), 2.11-2.29 (3H, m), 2.95-3.04 (1H, m), 3.21-3.38(3H, m), 3.62 (2H, d, J=5.8Hz), 3.98-4.07 (1H, m), 4.13-4.20 (1H, m),6.74 (1H, s), 7.01 (1H, d, J=15.4Hz), 7.56 (1H, dd, J=2.0, 7.3Hz), 7.58(1H, d, J=15.4Hz), 7.99-8.02 (1H, m), 8.93 (1H, dd, J=0.7, 7.3Hz).

IR (ATR) cm⁻¹: 3321, 2929, 2854, 1664, 1639, 1516, 1439, 1365, 1311,1282, 1234, 1101, 1061, 1003.

m.p.: 167-175° C.

ES-MS: m/z: 583 (MH⁺).

Example 266{2-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetylamino]ethyl}-(2-hydroxyethyl)dimethylammonium

(A)(2-{2-[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]acetylamino}ethyl)-(2-hydroxyethyl)dimethylammonium

A solution of2-{3-[(2-dimethylaminoethylaminocarbonyl)methyl]piperidin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide (113 mg) in N,N-dimethylformamide(2 ml) was added with iodoethanol (12.9 μl) and stirred at roomtemperature for 19 hours. The reaction mixture was added withiodoethanol (12.9 μl ) and stirred at room temperature 10 hours and 30minutes. The reaction mixture was added with iodoethanol (58.5 μl) andstirred at room temperature for 20 hours. The reaction mixture was addedwith iodoethanol (58.5 μl) and stirred at room temperature for 25 hours.The reaction mixture was added with saturated aqueous sodiumhydrogencarbonate (1 ml), stirred at room temperature for 30 minutes,then added with iodoethanol (1 ml) and stirred at room temperature for20 hours. After further stirred for 22 hours and 30 minutes, thereaction mixture was subjected concentrated under reduced pressure byazeotropy with N,N-dimethylformamide and toluene, and the residue waspurified by preparative TLC (4 plates,chloroform:methanol:water=8:3:0.5) to obtain the title compound (141 mg,quantitative) as orange solid.

¹H-NMR (CD₃OD-CDCl₃) δ: 1.31-1.37 (1H, m), 1.35 (9H, s), 1.63-1.85 (2H,m), 1.91-1.99 (1H, m), 2.13-2.26 (3H, m), 2.91-3.01 (1H, m), 3.11-3.17(1H, m), 3.23 (6H, s), 3.51-3.57 (4H, m), 3.59-3.65 (2H, m), 3.76 (3H,s), 3.98-4.09 (4H, m), 5.60 (2H, s), 6.73 (1H, m), 6.78 (2H, d,J=8.8Hz), 7.33 (2H, d, J=8.8Hz), 7.55-7.59 (1H, m), 7.63 (1H, d,J=15.6Hz), 7.76 (1H, d, J=15.6Hz), 7.94 (1H, d, J=1.5Hz), 8.93 (1H, d,J=7.8Hz).

(B){2-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-3-yl)acetylamino]ethyl}-(2-hydroxyethyl)dimethylammonium

(2-{2-[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-3-yl]acetylamino}ethyl)-(2-hydroxyethyl)dimethylammonium(141 mg) was dissolved in anisole (1 ml) and trifluoroacetic acid (2 ml)and stirred at 80° C. for 4 hours. The reaction mixture was concentratedunder reduced pressure by azeotropy with toluene, and the residue waspurified by preparative TLC (3 plates,chloroform:methanol:water=8:3:0.5) and further purified by HPLC toobtain the title compound (21.9 mg) as orange solid.

¹H-NMR (CD₃OD) δ: 1.22-1.39 (1H, m), 1.34 (9H, s), 1.64-1.95 (3H, m),2.02-2.24 (3H, m), 2.81-2.93 (1H, m), 3.01-3.15 (1H, m), 3.17 (6H, s),3.32-3.51 (6H, m), 3.88-4.03 (3H, m), 4.07-4.19 (1H, m), 6.70 (1H, s),7.33 (1H, d, J=16.4Hz), 7.45-7.56 (1H, m), 7.83-7.94 (2H, m), 8.88 (1H,d, J=7.3Hz).

IR (ATR) cm⁻¹: 3219, 2958, 2927, 2852, 1655, 1512, 1421, 1373, 1308,1252, 1227, 1101, 1066.

ESI-MS m/z: 678 (M⁺).

m.p.: 207-217° C.

Example 267{2-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-4-yl)acetylamino]ethyl}aminocarbonylmethyldimethylammonium

(A) 4-Ethoxycarbonylmethylene-piperidine-1-tert-butyl ester

A solution of ethyl diethylphosphonoacetate (11.1 ml) in tetrahydrofuran(100 ml) was added with sodium hydride (95%, 1.32 g) at 0° C. andstirred at room temperature for 15 minutes. The reaction mixture wasadded with a solution of 4-oxo-piperidine-1-tert-butyl ester (10.0 g) intetrahydrofuran (100 ml) at 0° C. and stirred at room temperature for 14hours. The reaction mixture was diluted with water and extracted withethyl acetate, and the organic layer was dried over anhydrous magnesiumsulfate. The solvent was concentrated under reduced pressure, and theresidue was purified by silica gel column chromatography (500 g,hexane:ethyl acetate=7:1) to obtain the title compound (12.2 g, 90%) aswhite solid.

¹H-NMR (CDCl₃) δ: 1.28 (3H, t, J=7.1Hz), 1.47 (9H, s), 2.28 (2H, t,J=5.7Hz), 2.93 (2H, t, J=5.7Hz), 4.15 (2H, t, J=5.7Hz), 4.16 (2H, t,J=5.7Hz), 5.71 (1H, s).

(B) 4-Ethoxycarbonylmethylpiperidine-1-tert-butyl ester

A solution of 4-ethoxycarbonylmethylenepiperidine-1-tert-butyl ester(7.17 g) in ethanol (100 ml) was added with 5% Pd-C (wet, 1.5 g) andstirred at room temperature for 17 hours under hydrogen atmosphere. Thecatalyst was removed by filtration through a Celite layer and washedwith ethanol, and the filtrate and the washing solution were combinedand concentrated. The residue was removed by filtration through a silicagel pad and eluted with ethyl acetate. The solvent was concentratedunder reduced pressure to obtain the title compound (7.31 g,quantitative) as colorless oily substance.

¹H-NMR (CDCl₃) δ: 1.16 (2H, dq, J=3.7, 12.5Hz), 1.26 (3H, t, J=7.1Hz),1.45 (9H, s), 1.69 (2H, br.d, J=11.0Hz), 1.87-1.99 (1H, m), 2.23 (2H, d,J=7.1Hz), 2.72 (2H, br.t, J=12.5Hz), 4.00-4.17 (2H, m), 4.13 (2H, q,J=7.1Hz).

(C) Piperidin-4-yl-acetic acid ethyl ester hydrochloride

4-Ethoxycarbonylmethylpiperidine-1-tert-butyl ester (7.31 g) wasdissolved in 4 N solution of hydrochloric acid in dioxane (70 ml) andstirred at room temperature for 2 hours. The solvent was evaporated byazeotropy with toluene to obtain the title compound (6.16 g,quantitative) as a mixture of colorless solid and oily substance.

¹H-NMR (CDCl₃) δ: 1.26 (3H, t, J=7.1Hz), 1.64-1.80 (2H, m), 1.85-2.11(3H, m), 2.31 (2H, d, J=7.1Hz), 2.81-2.97 (2H, m), 3.72 (2H, d,J=7.1Hz), 4.14 (2H, q, J=7.1Hz).

(D)[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-4-yl]aceticacid ethyl ester

The following reaction was performed under argon atmosphere. A solutionof2-hydroxy-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-(4-tert-butylthiazol-2-yl)amide(500 mg) in N,N-dimethylformamide (4 ml) was added withdiisopropylethylamine (624 μl) and diphenylphosphoryl chloride (371 μl)at 0° C. and stirred at 0° C. for 30 minutes. The reaction mixture wasadded with piperidin-4-yl-acetic acid ethyl ester hydrochloride (372 mg)and diisopropylethylamine (312 μl) at the same temperature and stirredat 80° C. for 1 hour. The reaction mixture was further added withpiperidin-4-yl-acetic acid ethyl ester hydrochloride (372 mg) anddiisopropylethylamine (312 μl) at the same temperature and stirred at80° C. for 2 hours. The reaction mixture was returned to roomtemperature, diluted with water and extracted with ethyl acetate. Theorganic layer was dried over anhydrous magnesium sulfate, and thesolvent was concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (50 g, chloroform:ethylacetate=4:1) and silica gel column chromatography (40 g, hexane:ethylacetate=2:1→3:2→1:1) to obtain the title compound (233 mg) as a hardlyseparable mixture.

¹H-NMR (CDCl₃) δ: 1.26 (3H, t, J=7.1Hz), 1.37 (9H, s), 1.39-1.50 (2H,m), 1.83-(2H, br.d, J=12.5Hz), 2.03-2.16 (1H, m), 2.31 (2H, d, J=7.1Hz),3.07-3.19 (2H, m), 3.80 (3H, s), 4.08-4.13 (2H, m), 4.15 (2H, q,J=7.1Hz), 5.59 (3H, s), 6.65 (1H, s), 7.22 (2H, d, J=8.8Hz), 7.36 (2H,d, J=8.8Hz), 7.58 (1H, dd, J=1.8, 7.3Hz), 7.66 (1H, d, J=15.6Hz), 7.85(1H, d, J=15.6Hz), 8.00-8.04 (1H, m), 8.94 (1H, d, J=7.3Hz).

(E)[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-4-yl]aceticacid

A solution of[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-4-yl]aceticacid ethyl ester (233 mg) in tetrahydrofuran (2 ml) was added with 1 Naqueous sodium hydroxide (655 μl) and stirred at 50° C. for 1 hour. Thenthe reaction mixture was added with 1 N aqueous sodium hydroxide (327μl) four times with stirring at 50° C. over 6 hours and 30 minutes. Thereaction mixture was returned to room temperature and adjusted to aboutpH 4 by adding 1 N hydrochloric acid. The reaction mixture was dilutedwith saturated brine and extracted with chloroform. The organic layerwas dried over anhydrous magnesium sulfate. The solvent was concentratedunder reduced pressure to obtain the title compound (223 mg, 36% for thetwo steps) as orange foamy syrup.

¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 1.55-1.70 (2H, m), 1.91-2.01 (2H, m),2.21-2.35 (1H, m), 2.51-2.60 (2H, m), 3.00-3.13 (2H, m), 3.76 (3H, s),3.95-4.04 (2H, m), 5.49 (2H, s), 6.64 (1H, s), 6.85 (2H, d, J=8.8Hz),7.31 (2H, d, J=8.8Hz), 7.55 (1H, dd, J=1.5, 7.3Hz), 7.70 (1H, d,J=15.5Hz), 7.89 (1H, d, J=15.5Hz), 8.28 (1H, d, J1.5Hz), 8.98 (1H, d,J=7.3Hz).

(F)2-{4-[(2-Dimethylaminoethylaminocarbonyl)methyl]piperidin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-8-(4-tert-butylthiazol-2-yl)amide

A solution of[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-4-yl]aceticacid (223 mg) and N,N-dimethylethylenediamine (35.8 μl) in methylenechloride (3 ml) and N,N-dimethylformamide (1 ml) was added withtriethylamine (114 μl), HOBt (48.5 mg) and WSCD.HCl (68.8 mg) with icecooling and stirred at room temperature for 24 hours. The solvent wassubjected to azeotropy with toluene, and the residue was diluted withsaturated aqueous sodium hydrogencarbonate and extracted withchloroform. The organic layer was dried over anhydrous magnesiumsulfate, and the solvent was concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography (15 g,chloroform→chloroform:methanol=10:1) to obtain the title compound (185mg, 75%) as orange foamy syrup.

¹H-NMR (CDCl₃) δ: 1.33 (9H, s), 1.34-1.48 (2H, m), 1.70-1.80 (2H, m),1.97-2.09 (1H, m), 2.15-2.21 (2H, m), 2.30 (6H, s), 2.44-2.53 (2H, m),2.99 (2H, s), 2.95-3.08 (2H, m), 3.28-3.38 (2H, m), 3.72 (3H, s),4.00-4.11 (2H, m), 5.53 (2H, s), 6.64 (1H, s), 6.87 (2H, d, J=8.7Hz),7.29 (2H, d, J=8.7Hz), 7.45 (1H, dd, J=1.8, 7.3Hz), 7.52 (1H, d,J=15.6Hz), 7.71 (1H, d, J=15.6Hz), 7.80-7.84 (1H, m), 8.78 (1H, d,J=7.3Hz).

(G)(2-{2-[1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-4-yl]acetylamino}ethyl)aminocarbonylmethyldimethylammonium

A solution of2-{4-[(2-dimethylaminoethylaminocarbonyl)methyl]piperidin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-8-(4-tert-butylthiazol-2-yl)amide(83.3 mg) in N,N-dimethylformamide (2 ml) was added with iodoacetamide(22.5 mg) and stirred for 16 hours with light shielding. The reactionmixture was further added with iodoacetamide (11.0 mg) and stirred for24 hours with light shielding. The solvent was evaporated by azeotropywith toluene, and the residue was purified by preparative TLC (2 plates,chloroform:methanol:water=8:3:0.5) to obtain the title compound (102 mg,98%) as orange solid.

¹H-NMR (CD₃OD) δ: 1.34 (9H, s), 1.40-1.54 (2H, m), 1.75-1.86 (2H, m),2.00-2.29 (3H, m), 3.02-3.15 (2H, m), 3.37 (6H, s), 3.67-3.82 (4H, m),3.76 (3H, s), 4.09-4.23 (4H, m), 5.59 (2H, s), 6.72 (1H, s), 6.91 (2H,d, J=8.8Hz), 7.33 (2H, d, J=8.8Hz), 7.51-7.58 (1H, m), 7.61 (1H, d,J=15.6Hz), 7.80 (1H, d, J=15.6Hz), 7.91-7.96 (1H, m), 8.88-8.95 (1H, m).

(H){2-[2-(1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-4-yl)acetylamino]ethyl}aminocarbonylmethyldimethylammonium

A solution of(2-{2-[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-4-yl]acetylamino}ethyl)aminocarbonylmethyldimethylammonium(102 mg) in trifluoroacetic acid (2 ml) was stirred at 50° C. for 4hours and at 80° C. for 4 hours. The solvent was evaporated by azeotropywith toluene, and the residue was purified by preparative TLC (2 plates,chloroform:methanol:water=8:3:0.5, developed twice) to obtain the titlecompound (62.7 mg) as red solid.

Anal. Calcd. for C₃₂H₄₂N₁₂O₄S.3H₂O.3.5TFA: C, 40.95; H, 4.54; N, 14.69;S, 2.80. Found: C, 40.83; H, 4.35; N, 14.91; S, 2.91.

¹H-NMR (400 MHz, CD₃OD) δ: 1.25 (9H, s), 1.35-1.49 (2H, m), 1.64-1.75(2H, m), 1.90-2.02 (1H, m), 2.10-2.18 (2H, m), 2.90-3.01 (2H, m), 3.23(6H, s), 3.53-3.68 (4H, m), 4.05 (2H, s), 4.04-4.16 (2H, m), 6.65 (1H,s), 7.31 (1H, d, J=16.4Hz), 7.49 (1H, dd, J=2.0, 7.6Hz), 7.83 (1H, d,J=16.4Hz), 7.89-7.91 (1H, m), 8.89 (1H, d, J=7.6Hz).

IR (ATR) cm⁻¹: 3317, 2964, 2862, 1670, 1518, 1425, 1373, 1311, 1203,1176, 1126, 1072, 1030.

ES-MS: m/z: 691 (M⁺).

Anal. Calcd. for C₃₂H₄₂N₁₂O₄S.3H₂O.3.5TFA: C, 40.95; H, 4.54; N, 14.69;S, 2.80.found: C, 40.83; H, 4.35; N, 14.91; S, 2.91.

m.p.: 151-158° C.

Example 268(2-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl]piperidin-4-yl)acetylamino]ethyl}carboxymethyldimethylammonium

(A)tert-Butoxycarbonylmethyl-(2-{2-[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-4-yl]acetylamino}ethyl)dimethylammonium

A solution of2-{3-[(2-dimethylaminoethylaminocarbonyl)methyl]piperidin-1-yl}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxylicacid (4-tert-butylthiazol-2-yl)amide (87.5 mg) in N,N-dimethylformamide(2 ml) was added with bromoacetic acid tert-butyl ester (18.9 μl) andstirred for 16 hours with light shielding. The reaction mixture wasadded with bromoacetic acid tert-butyl ester (18.9 μl), stirred for 31hours-with tight shielding, further added with bromoacetic acidtert-butyl ester (18.9 μl) and stirred for 3.5 days with lightshielding. The solvent was evaporated by azeotropy with toluene, and theresidue was purified by preparative TLC (2 plates,chloroform:methanol:water=8:3:0.5) to obtain the title compound (121 mg)as orange solid.

¹H-NMR (CDCl₃) δ: 1.30-1.55 (2H, m), 1.37 (9H, s), 1.47 (9H, s),1.70-1.83 (2H, m), 2.06-2.20 (1H, m), 2.25-2.40 (2H, m), 2.87-3.04 (2H,m), 3.66 (6H, s), 3.72-3.87 (2H, s), 3.77 (3H, s), 4.00-4.15 (4H, m),4.67 (2H, s), 5.53 (2H, s), 6.66 (1H, s), 6.90 (2H, d, J=8.4Hz),7.25-7.41 (1H, m), 7.36 (2H, d, J=8.4Hz), 7.36 (1H, d, J=15.4Hz), 7.79(1H, br), 7.90 (1H, d, J=15.4Hz), 8.72-8.86 (2H, m).

(B){2-[2-(1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-tetrazol-5-yl)vinyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}piperidin-4-yl)acetylamino]ethyl}carboxymethyldimethylammonium

tert-Butoxycarbonylmethyl-(2-{2-[1-(8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-tetrazol-5-yl]vinyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)piperidin-4-yl]acetylamino}ethyl)dimethylammonium(121 mg) was dissolved in 4 N hydrochloric acid in dioxane (2 ml) andstirred at room temperature 13 hours and 30 minutes. The solvent wasevaporated by azeotropy with chloroform, and the residue was dissolvedin trifluoroacetic acid (2 ml) and stirred at 80° C. for 3 hours. Thesolvent was evaporated by azeotropy with toluene, and the residue waspurified by preparative TLC (3 plates,chloroform:methanol:water=8:3:0.5) to obtain the title compound (121 mg)as red solid.

¹H-NMR (CD₃OD) δ: 1.25 (9H, s), 1.32-1.45 (2H, m), 1.65-1.75 (2H, m),1.91-2.04 (1H, m), 2.08-2.16 (2H, m), 2.97-3.07 (2H, m), 3.19 (6H, s),3.50-3.57 (2H, m), 3.60-3.68 (2H, m), 3.76 (2H,s), 4.08-4.17 (2H, m),6.64 (1H, s), 7.38 (1H, d, J=16.1Hz), 7.48 (1H, dd, J=1.8, 7.6Hz), 7.81(1H, dd, J=16.1Hz), 7.89-7.92 (1H, m), 8.88 (1H, d, J=7.6Hz).

IR (ATR) cm⁻¹: 3332, 2964, 2866, 1657, 1635, 1516, 1439, 1404, 1373,1348, 1203, 1178, 1128, 1070.

ES-MS m/z: 692 (MH)⁺.

Anal. Calcd. for C₃₂H₁₄N₁₁O₅S.3.5TFA.5H₂O: C, 39.66; H, 4.65; N, 13.05;S, 2.72. Found: C, 39.27; H, 4.24; N, 13.33; S, 2.72.

m.p.: 148-158° C.

Example 269(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{4-[(2-hydroxyethylaminocarbonyl)methyl]piperidin-1-yl}-4-oxo-4H-pyrido[1,2-a]-pyrimidin-3-yl)-2-propenoicacid

(A)(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-ethoxycarbonylmethylpiperidin-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid tert-butyl ester

The following reaction was performed under argon atmosphere. A solutionof(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid tert-butyl ester (500 mg) in N,N-dimethylformamide (4 ml) was addedwith diisopropylethylamine (740 μl) and diphenylphosphoryl chloride (441μl) at 0° C. and stirred at 0° C. for 30 minutes. The reaction mixturewas added with piperidin-4-yl-acetic acid ethyl ester hydrochloride (441mg) and diisopropylethylamine (740 μl) at the same temperature andstirred at 80° C. for 1 hour. The reaction mixture was returned to roomtemperature, diluted with water and extracted with chloroform. Theorganic layer was dried over anhydrous magnesium sulfate, and thesolvent was concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (35 g,chloroform→chloroform:ethyl acetate=7:1, and 25g, hexane:ethylacetate=3:1) to obtain the title compound (494 mg) as a hardly separablemixture.

(B)(E)-3-[8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-carboxymethylpiperidin-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid tert-butyl ester

A solution of(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-ethoxycarbonylmethylpiperidin-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid tert-butyl ester (494 mg) in tetrahydrofuran (2 ml) was added with1 N aqueous sodium hydroxide (2.38 ml) and stirred at 50° C. for 1 hour.Then the reaction mixture was added with 1 N aqueous sodium hydroxide(2.38 ml) four times with stirring at 50° C. over 5 hours. The reactionmixture was returned to room temperature, then added with 1 Nhydrochloric acid and adjusted to about pH 4. The reaction mixture wasdiluted with saturated brine and extracted with chloroform. The organiclayer was dried over anhydrous magnesium sulfate, and the solvent wasconcentrated under reduced pressure. The residue was purified by silicagel column chromatography (20 g, hexane:ethyl acetate=1:1→ethyl acetate)to obtain the title compound (241 mg, 38% for the two steps) as orangesolid.

¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 1.48 (9H, s), 1.49-1.63 (2H, m),1.94-2.02 (2H, m), 2.23-2.35 (1H, m), 2.51-2.58 (2H, m), 3.01-3.11 (2H,m), 3.95-4.04 (2H, m), 6.65 (1H, s), 7.13 (1H, d, J=15.7Hz), 7.44 (1H,d, J=15.7Hz), 7.54 (1H, dd, J=1.7, 7.3Hz), 8.25 (1H, d, J=1.7Hz), 9.01(1H, d, J=7.3Hz).

(C)(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{4-[(2-hydroxyethylaminocarbonyl)methyl]piperidin-1-yl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid tert-butyl ester

A solution of(E)-3-[8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-(4-carboxymethylpiperidin-1-yl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]-2-propenoicacid tert-butyl ester (120 mg) and ethanolamine (12.2 μl) in methylenechloride (2 ml) and N,N-dimethylformamide (2 ml) was added withtriethylamine (70.2 μl), HOBt (29.9 mg) and WSCD.HCl (42.5 mg) with icecooling and stirred at room temperature for 18 hours. The solvent wassubjected to azeotropy with toluene, and the residue was diluted withchloroform and washed with 10% aqueous citric acid and saturated aqueoussodium hydrogencarbonate. The organic layer was dried over anhydrousmagnesium sulfate, and the solvent was concentrated under reducedpressure. The residue was purified by preparative TLC (3 plates,chloroform:methanol=10:1) to obtain the title compound (98.3 mg, 76%) asorange foamy syrup.

¹H-NMR (CDCl₃) δ: 1.28-1.44 (2H, m), 1.34 (9H, s), 1.51 (9H, s),1.76-2.00 (3H, m), 2.07-2.24 (3H, m), 2.93-3.04 (2H, m), 3.44-3.54 (2H,m), 3.79 (2H, t, J=4.9Hz), 4.04-4.12 (2H, m), 6.52-6.60 (1H, m), 6.58(1H, s), 7.01 (1H, d, J=15.6Hz), 7.35 (1H, d, J=15.6Hz), 7.43 (1H, dd,J=1.7, 7.4Hz), 7.86-7.91 (1H, m), 8.92 (1H, d, J=7.4Hz).

(D)(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{4-[(2-hydroxyethylaminocarbonyl)methyl]piperidin-1-yl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid

(E)-3-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-2-{4-[(2-hydroxyethylaminocarbonyl)methyl]piperidin-1-yl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)-2-propenoicacid tert-butyl ester (98.3 mg) was dissolved in 4 N hydrochloric acidin dioxane (2 ml) and stirred at room temperature for 12 hours and 30minutes. As the reaction was not completed, the solvent was concentratedby azeotropy with toluene. The residue was dissolved in 4 N hydrochloricacid in dioxane (4 ml) and stirred at room temperature for 1 hour. Asthe reaction did not proceed, the solvent was concentrated by azeotropywith toluene, and the residue was dissolved in trifluoroacetic acid (2ml) and stirred at room temperature for 1 hour and 30 minutes. Thesolvent was concentrated by azeotropy with toluene, and the residue waspurified by preparative TLC (3 plates,chloroform:methanol:water=8:3:0.5) to obtain the title compound (67.5mg) as orange solid.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.38-1.52 (2H, m), 1.78-1.89 (2H, m),2.03-2.25 (3H, m), 3.08-3.20 (2H, m), 3.28-3.33 (2H, m), 3.60 (2H, t,J=5.9Hz), 4.16-4.24 (2H, m), 6.74 (1H, s), 7.03 (1H, d, J=15.5Hz), 7.56(1H, dd, J=1.8, 7.3Hz), 7.58 (1H, d, J=15.5Hz), 7.99-8.00 (1H, m), 8.94(1H, dd, J=0.7, 7.3Hz).

IR (ATR) cm⁻¹: 3323, 2929, 2868, 2848, 1658, 1514, 1442, 1427, 1365,1313, 1282, 1225, 1190, 1101, 1063, 1005.

ES-MS: m/z: 583 (MH⁺).

Anal. Calcd. for C₂₈H₃₄N₆O₆S.1.75H₂O: C, 54.75; H, 6.15; N, 13.68; S,5.22. Found: C, 54.54; H, 5.66; N, 13.43; S, 5.16.

m.p.: 173-183° C.

Example 270[2-([((3R)-1-{8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl-hexahydro-3-pyridinyl}oxy)carbamoyl]amino)ethyl](carboxymethyl)dimethylammonium

(A)[2-([((3R)-1-{8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-4-oxo-3-[(E)-2-{1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-ylhexahydro-3-pyridinyl}oxy)carbamoyl]amino)ethyl](carboxymethyl)dimethylammonium

(3R)-1-(8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxycarbonyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate (405 mg, 0.536 mmol) was dissolvedin N,N-dimethylformamide (8 ml), added with t-butyl bromoacetate (0.396ml, 2.67 mmol) and stirred overnight at room temperature. The reactionmixture was concentrated under reduced pressure. After completion of thereaction was confirmed, the resulting residue was dissolved in 4 Nhydrochloric acid in dioxane (10 ml) and stirred at room temperature for9 hours. The reaction mixture was concentrated under reduced pressure,and the resulting residue was purified by preparative TLC(chloroform:methanol:water=8:3:1) to obtain the title compound (329 mg,72%).

¹H-NMR (CD₃OD) δ: 1.32 (9H, s), 1.60 (1H, m), 1.88 (3H, m), 2.03 (1H,m), 3.23-3.26 (8H, m), 3.52-3.60 (4H, m), 3.67-3.74 (6H, m), 3.82-3.85(2H, m), 5.52 (2H, m), 6.64 (1H, m), 6.86 (2H, m), 7.27 (2H, m), 7.47(1H, m), 7.65 (2H, m), 7.82 (1H, m), 8.78 (1H, m).

ES-MS: m/z: 815 (M⁺)

(B)[2-([((3R)-1-{8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-ylhexahydro-3-pyridinyl}oxy)carbamoyl]amino)ethyl](carboxymethyl)dimethylammonium

[2-([((3R)-1-{8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-4-oxo-3-[(E)-2-{1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl-hexahydro-3-pyridinyl}oxy)carbamoyl]amino)ethyl](carboxymethyl)dimethylammonium(329 mg, 0.387 mmol) was dissolved in trifluoroacetic acid (10 ml),added with anisole (three drops) and stirred at 60° C. for 2 hours. Thereaction mixture was left stand for cooling, concentrated under reducedpressure and subjected to azeotropy with toluene several times. Theresulting residue was purified by preparative TLC(chloroform:methanol:water=8:3:1, developed three times) and eluted fromsilica gel with chloroform:methanol:water=6:4:1 to obtaintrifluoroacetic acid salt of the title compound (328 mg). This compoundwas dissolved in methanol/water, and the solution was adjusted to pH'8with 0.1 N aqueous sodium hydroxide, purified by HPLC and lyophilized toobtain the title compound (103 mg, 38%).

HPLC

Column: SHISEIDO CAP CELL PAK C18, SG-120A, 30×250 mm

Elute: methanol:water=60:40

FR: 8 ml/min

Retention time: 23 min.

¹H-NMR (DMSO-d₆) δ: 1.30 (9H, s), 1.69 (1H, m), 1.85 (2H, m), 2.00 (1H,m), 3.20 (6H, s), 3.45 (2H, m), 3.61-3.69 (8H, m), 4.69 (1H, br), 6.71(1H, brs), 7.55 (1H, d, J=15.9Hz), 7.70 (1H, d, J=7.1Hz), 7.99 (1H, d,J=15.-9Hz), 8.17 (1H, m), 8.78 (1H, br), 8.96 (1H, d, J=7.6Hz).

¹H-NMR ((CD₃OD) δ: 1.29 (9H, s), 1.69 (1H, m), 1.94 (2H, m), 2.03 (1H,m), 3.26 (6H, s), 3.44-3.71 (6H, m), 3.84 (2H, s), 3.92 (1H, m), 4.06(1H, m), 6.71 (1H, s), 7.45 (1H, d, J=15.9Hz), 7.64 (1H, dd, J=1.7and7.6Hz), 7.94 (1H, d, J=15.9Hz), 8.04 (1H, d, J=1.7Hz), 8.96 (1H, d,J=7.6Hz)

IR (ATR) cm⁻¹: 2958, 1700, 1650, 1625, 1511, 1423, 1247, 1103.

m.p.: 220-233° C. (decomp.)

ES-MS: m/z: 694 (M⁺)

Anal. Calcd. for C₃₁H₃₉N₁₁O₆S.6H₂O: C, 46.43; H, 6.41; N, 19.21. Found:C, 46.48; H, 5.86; N, 18.66.

Example 271[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2-fluoroethyl)dimethylammonium

(A)[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-{1-(4-methoxycarbonyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2-fluoroethyl)dimethylammonium

(3R)-1-(8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxycarbonyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate (323 mg, 0.427 mmol) was dissolvedin N,N-dimethylformimidazole-4-yl (6 ml), added with1-bromo-2-fluoroethane (0.159 ml, 2.14 mmol) and stirred overnight atroom temperature. The reaction mixture was further added with1-bromo-2-fluoroethane (0.159 ml, 2.14 mmol) and stirred at 80° C. for 2days. The reaction mixture was concentrated under reduced pressure, andthe resulting residue was purified by preparative TLC(chloroform:methanol:water=8:3:1) to obtain the title compound (322 mg,85%).

¹H-NMR (CD₃OD) δ: 1.22 (9H, s), 1.53 (1H, m), 1.79 (3H, m), 3.16 (6H,s), 3.11-3.24 (4H, m), 3.42-3.66 (2H, m), 3.62 (3H, s), 3.78 (2H, m),4.87 (2H, d, J=49.3Hz), 5.41 (2H, s), 6.54 (1H, s), 6.77 (2H, d,J=8.5Hz), 7.18 (2H, d, J=8.5Hz), 7.39 (1H, m), 7.55 (2H, m), 7.69 (1H,m), 8.55 (1H, m).

ES-MS: m/z: 802 (M⁺)

(B)[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2-fluoroethyl)dimethylammonium

[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-{1-(4-methoxycarbonyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonylamino)ethyl](2-fluoroethyl)dimethylammonium(322 mg, 0.365 mmol) was dissolved in trifluoroacetic acid (10 ml),added with anisole (5 drops) and stirred at 60° C. for 5 hours. Thereaction mixture was left stand for cooling, concentrated under reducedpressure and subjected to azeotropy with toluene several times. Theresulting residue was purified by preparative TLC(chloroform:methanol:water=8:3:1, developed three times) and eluted fromsilica gel with chloroform:methanol:water=6:4:1 to obtaintrifluoroacetic acid salt of the title compound (145 mg). This compoundwas dissolved in methanol/water, and the solution was adjusted to pH'8with 0.1 N aqueous sodium hydroxide, purified by HPLC and lyophilized toobtain the title compound (98.0 mg, 39%).

HPLC

Column: SHISEIDO CAPCELL PAK C18, SG-120A, 30×250 mm

Elute: methanol:water=70:30

FR: 12 ml/min

Retention time: 32 min.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.71 (1H, m), 1.92-2.01 (3H, m), 3.22(6H, s), 3.40 (1H, m), 3.51-3.60 (5H, m), 3.79 (1H, m), 3.88-3.95 (3H,m), 4.95 (2H, d, J=47.0Hz), 6.74 (1H, s), 7.56 (1H, d, J=16.1Hz), 7.60(1H, dd, J=1.7and 7.6Hz), 7.96 (1H, d, J=16.1Hz), 8.01 (1H, d, J=1.7Hz),8.98 (1H, d, J=7.6Hz)

IR (ATR) cm⁻¹: 2958, 1658, 1513, 1425, 1249, 1103.

ES-MS: m/z: 682 (MH⁺), 680 (MH⁻)

Anal. Calcd. for C₃₁H₄₀N₁₁O₄FS.3H₂O: C, 50.60; H, 6.30; N, 20.94; F,2.58. Found: C, 51.13; H, 6.24; N, 20.21; F, 2.62.

Example 272[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2-hydroxyethyl)dimethylammonium

(A)[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-{1-(4-methoxycarbonyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2-hydroxyethyl)dimethylammonium

(3R)-1-(8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxycarbonyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate (331 mg, 0.438 mmol) was dissolvedin N,N-dimethylformamide (3 ml), added with 2-iodoethanol (0.171 ml,2.19 mmol) and stirred overnight at room temperature. The reactionmixture was further added with 2-iodoethanol (0.171 ml, 2.19 mmol) andstirred at 80° C. for 2 days. The reaction mixture was concentratedunder reduced pressure, and the resulting residue was purified bypreparative TLC (chloroform:methanol:water=8:3:1) to obtain the titlecompound (465 mg, quantitative).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.70 (1H, m), 1.95 (3H, m), 3.23 (6H,s), 3.54-3.71 (10H, m), 3.62 (3H, s), 5.41 (2H, s), 6.72 (1H, s), 6.92(2H, d, J=8.7Hz), 7.32 (2H, d, J=8.7Hz), 7.61 (1H, m), 7.80 (2H, m),7.97 (1H, m), 8.95 (1H, m).

ES-MS: m/z: 800 (M⁺)

(B)[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2-hydroxyethyl)dimethylammonium

[2-({[((3R)-1-{8-({[4-(-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-{1-(4-methoxycarbonyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2-hydroxyethyl)dimethylammonium (465 mg, 0.501 mmol) wasdissolved in trifluoroacetic acid (10 ml), added with anisole (threedrops) and stirred at 60° C. for 5 hours. The reaction mixture was leftstand for cooling, concentrated under reduced pressure and subjected toazeotropy with toluene several times. The resulting residue was purifiedby preparative TLC (chloroform:methanol:water=8:3:1, developed threetimes) and eluted from silica gel with chloroform:methanol:water=6:4:1to obtain trifluoroacetic acid salt of the title compound (190 mg). Thiscompound was dissolved in methanol/water, and the solution was adjustedto pH'8 with 0.1 N aqueous sodium hydroxide, purified by HPLC andlyophilized to obtain the title compound (107 mg, 31%).

HPLC

Column: SHISEIDO CAPCELL PAK C18, SG-120A, 30×250 mm

Elute: methanol:water=70:30

FR: 12 ml/min

Retention time: 28 min.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.70 (1H, m), 1.90-2.01 (3H, m), 3.20(6H, s), 3.41-3.63 (8H, m), 3.90 (2H, m), 4.00 (2H, m), 6.73 (1H, s),7.55 (1H, d, J=16.1Hz), 7.59 (1H, dd, J=1.5and 7.6Hz), 7.93 (1H, d,J=16.1Hz), 8.00.(1H, d, J=1.5Hz), 8.97 (1H, d, J=7.6Hz)

IR (ATR) cm⁻¹: 3218, 2958, 1704, 1658, 1540, 1513, 1425, 1247, 1226

ES-MS: m/z: 680 (M⁺), 679 (MH⁻)

Anal. Calcd. for C₃₁H₄₁N₁₁O₅S.3.25H₂O: C, 50.43; H, 6.48; N, 20.87.Found: C, 51.18; H, 6.28; N, 19.96.

Example 273[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2,2-difluoroethyl)dimethylammonium

(A)[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-{1-(4-methoxycarbonyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2,2-difluoroethyl)dimethylammonium

(3R)-1-(8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxycarbonyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[2-(dimethylamino)ethyl]carbamate (343 mg, 0.454 mmol) was dissolvedin N,N-dimethylformamide (3 ml), added with 2-bromo-1,1-difluoroethane(0.180 ml, 2.27 mmol) and stirred overnight at room temperature. Thereaction mixture was added with 2-bromo-1,1-difluoroethane (0.180 ml,2.27 mmol) and stirred at 80° C. for 9 days. The reaction mixture wasconcentrated under reduced pressure, and the resulting residue waspurified by preparative TLC (chloroform:methanol:water=8:3:1) to obtainthe title compound (210 mg, 51%).

¹H-NMR (CDCl₃) δ: 1.37 (9H, s), 1.57 (1H, m), 1.82 (3H, m), 3.18 (2H,m), 3.48 (3H, s), 3.72 (2H, m), 3.83 (2H, m), 4.04 (1H, m), 4.47 (1H,m), 4.73 (1H, m), 5.49 (2H, s), 6.56 (1H, s), 6.74 (1H, m), 6.90 (2H, d,J=8.3Hz), 7.35 (2H, d, J=8.3Hz), 7.41 (1H, m), 7.88 (3H, m), 8.84 (1H,m).

ES-MS: m/z: 820 (M⁺)

(B)[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2,2-difluoroethyl)dimethylammonium

[2-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-{1-(4-methoxycarbonyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)ethyl](2,2-difluoroethyl)dimethylammonium(210 mg, 0.501 mmol) was dissolved in trifluoroacetic acid (5 ml), addedwith anisole (5 drops) and stirred at 60° C. for 5 hours. The reactionmixture was left stand for cooling, concentrated under reduced pressureand subjected to azeotropy with toluene several times. The resultingresidue was purified by preparative TLC(chloroform:methanol:water=8:3:1, developed three times) and eluted fromsilica gel with chloroform:methanol:water=6:4:1 to obtaintrifluoroacetic acid salt of the title compound (134 mg). This compoundwas dissolved in methanol/water, and the solution was adjusted to pH'8with 0.1 N aqueous sodium hydroxide, purified by HPLC and lyophilized toobtain the title compound (48.3 mg, 30%).

HPLC

Column: SHISEIDO CAPCELL PAK C18, SG-120A, 30×250 mm

Elute: methanol:water=70:30

FR: 12 ml/min

Retention time: 35 min.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.71 (1H, m), 1.86 (1H, m), 2.02 (1H,m), 3.30 (6H, s), 3.45 (1H, m), 3.60 (5H, m), 3.95-4.15 (4H, m), 6.60(1H, t, J=49.5Hz), 6.74 (1H, s), 7.56 (1H, d, J=16.1Hz), 7.61 (1H, d,J=7.8Hz), 7.98 (1H, d, J=16.1Hz), 8.03 (1H, s), 8.99 (1H, d, J=7.8Hz)

IR (ATR) cm⁻¹: 2960; 1700, 1654, 1517, 1425, 1249

ES-MS: m/z: 700 (M⁺)

Anal. Calcd. for C₃₁H₃₉F₂N₁₁O₄S.1C₂H₅OH.2.75H₂O: C, 49.83; H, 6.40; N,19.37; F, 4.78. Found: C, 50.17; H, 5.98; N, 18.93; F, 4.74.

Example 2742-{2-[({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)methyl]-1-pyridinium-yl}acetate

(A)(3R)-1-(8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-(2-pyridylmethyl)carbamate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(808 mg, 1.26 mmol) was dissolved in methylene chloride (16 ml), addedwith 1,1′-carbonyldiimidazole (306 mg, 1.89 mmol) and stirred at roomtemperature for 1 hour. The reaction mixture was added with2-(aminomethyl)pyridine (0.260 ml, 2.52 mmol) and further stirredovernight. The reaction mixture was diluted with chloroform and washedwith 1 N hydrochloric acid, saturated aqueous sodium hydrogencarbonateand saturated brine, and the organic layer was dried over anhydrousmagnesium sulfate. The solvent was evaporated and concentrated, and theresulting residue was purified by silica gel column chromatography(chloroform:methanol=100:1) to obtain the title compound (813 mg, 83%).

¹H-NMR (CDCl₃) δ: 1.33 (9H, s), 1.97 (3H, m), 3.79 (3H, s), 4.42 (1H,m), 4.58 (2H, m), 4.92 (1H, m), 5.40 (1H, m), 5.53 (2H, m), 6.60 (1H,m), 6.89 (2H, m), 7.45 (1H, m), 7.52 (1H, m), 7.90 (3H, m), 8.50 (2H,m), 8.97 (1H, m).

ES-MS: m/z: 776 (MH⁺)

(B)2-{2-[({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)methyl]-1-pyridinium-yl}-acetate

(3R)-1-(8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-(2-pyridylmethyl)carbamate (813 mg, 1.05 mmol) was dissolved inN,N-dimethylformamide (10 ml), added with t-butyl bromoacetate (2.32 ml,15.7 mmol) and stirred overnight at room temperature. The reactionmixture was concentrated under reduced pressure, and the resultingresidue was dissolved in 4 N hydrochloric acid in dioxane (15 ml) andstirred overnight at room temperature. The reaction mixture wasconcentrated under reduced pressure, and the resulting residue waspurified by preparative TLC (chloroform:methanol:water=8:3:1) to obtainthe title compound (515 mg, 59%).

ES-MS: m/z: 834 (M⁺)

(C)2-{2-[({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)methyl]-1-pyridinium-yl}acetate

2-{2-[({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)methyl]-1-pyridiniumyl}acetate(515 mg, 0.618 mmol) was dissolved in trifluoroacetic acid (13 ml),added with anisole (0.2 ml) and stirred at 60° C. for 4 hours. Thereaction mixture was left stand for cooling, concentrated under reducedpressure and subjected to azeotropy with toluene several times. Theresulting residue was purified by preparative TLC(chloroform:methanol:water=8:3:1, develop three times) and eluted fromsilica gel with chloroform:methanol:water=6:4:1 to obtaintrifluoroacetic acid salt of the title compound (413 mg). This compoundwas dissolved in methanol/water, and the solution was adjusted to pH'8with 0.1 N aqueous sodium hydroxide, purified by HPLC and lyophilized toobtain the title compound (10.2 mg, 2%).

HPLC

Column: SHISEIDO CAPCELL PAK C18, SG-120A, 30×250 mm

Elute: methanol:water=60:40

FR: 12 ml/min

Retention time: 17 min.

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.94 (2H, m), 2.03 (2H, m), 3.45 (1H,m), 3.72 (1H, m), 3.92 (2H, m), 4.55 (1H, d, J=17.1Hz), 4.67 (1H, d,J=17.1Hz), 5.18 (1H, d, J=16.6Hz), 5.24 (1H, d, J=16.6Hz), 6.73 (1H, s),7.43 (1H, d, J=16.1Hz), 7.56 (1H, d, J=5.6Hz), 7.86-7.91 (3H, m), 7.99(1H, s), 8.46 (1H, m), 8.78 (1H, d, J=6.1Hz), 8.95 (1H, d, J=7.6Hz)

IR (ATR) cm⁻¹: 2925, 1708, 1654, 1511, 1427, 1365, 1249, 1222

ES-MS: m/z: 714 (M⁺)

Anal. Calcd. for C₃₃H₃₅N₁₁O₆S.2C₂H₅OH.4H₂O: C, 50.61; H, 6.31; N, 17.55.Found: C, 50.68; H, 5.76; N, 17.19.

Example 2752-(2-(2S)-[({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-hexahydro-3-pyridinyl)oxy]carbonyl}amino)methyl]-1-ethyltetrahydro-1H-1-pyrrolium-yl}acetate

(A)(3R)-1-8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-4-oxo-3-[(E)-2-11-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl-hexahydro-3-pyridinylN-[(2S)-1-ethyltetrahydro-1H-2-pyrrol-yl]methylcarbamate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[(3R)-3-hydroxyhexahydro-1-pyridinyl]3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(524 mg, 0.817 mmol) was dissolved in methylene chloride (10 ml), addedwith 1,1′-carbonyldiimidazole (199 mg, 1.22 mmol) and stirred at roomtemperature for 1 hour and 30 minutes. The reaction mixture was addedwith a solution of (S)-(−)-2-aminomethyl-1-ethylpyrrolidine (209 mg,1.63 mmol) in methylene chloride (1 ml) and further stirred overnight.The reaction mixture was diluted with chloroform and washed with 1 Nhydrochloric acid, saturated aqueous sodium hydrogencarbonate andsaturated brine, and the organic layer was dried over anhydrousmagnesium sulfate. The solvent was evaporated and concentrated, and theresulting residue was purified by silica gel column chromatography(chloroform:methanol=100:3→100:5→10:1) to obtain the title compound (353mg, 54%).

¹H-NMR (CDCl₃) δ: 1.10 (3H, m), 1.37 (9H, a), 1.64 (4H, m), 1.90 ((4H,m), 2.65 (1H, m), 2.84 (1H, m), 3.15 (3H, m), 3.31 (2H, m), 3.62 (2H,m), 3.77 (3H, 8), 4.81 (1H, m), 5.48 (2H, s), 5.84 (1H, m), 6.56 (1H,s), 6.88 (1H, d, J=8.5Hz), 7.45 (1H, m), 7.76-7.90 (3H, m), 8.88 (1H, d,J=7.1Hz)

ES-MS: m/z: 796 (MH⁺)

(B)2-{2-(2S)-[({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-{1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]-pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbamoyl)amino)methyl]-1-ethyltetrahydro-1H-1-pyrrolium-yl}acetate

(3R)1-{8-([4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl}-4-oxo-3-[(E)-2-{1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl-hexahydro-3-pyridinylN-[(2S)-1-ethyltetrahydro-1H-2-pyrrolyl]methylcarbamate (353 mg, 0.443mmol) was dissolved in N,N-dimethylformamide (6 ml), added with t-butylbromoacetate (0.327 ml, 2.22 mmol) and stirred overnight at roomtemperature. The reaction mixture was further added with t-butylbromoacetate (0.981 ml) and stirred for 2 days. The reaction mixture wasfurther added with t-butyl bromoacetate (0.981 ml) and stirred at 80° C.for 8 hours. The reaction mixture was concentrated under reducedpressure, and the resulting residue was dissolved in 4 N hydrochloricacid in dioxane (10 ml) and stirred overnight at room temperature. Thereaction mixture was concentrated under reduced pressure, and theresulting residue was purified by preparative TLC(chloroform:methanol=10:1) to obtain the title compound (109 mg, 29%)and the starting substance (180 mg).

ES-MS: m/z: 854 (M⁺)

(C)2-{2-(2S)-[({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-hexahydro-3-pyridinyl)oxy]carbonyl}amino)methyl]-1-ethyltetrahydro-1H-1-pyrrolium-yl}acetate

2-{2-(2S)-[({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-{1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbamoyl}amino)methyl]-1-ethyltetrahydro-1H-1-pyrrolium-yl}acetate(54.2 mg, 0.0635 mmol) was dissolved in trifluoroacetic acid (2 ml),added with anisole (one drop) and stirred at 60° C. for 4 hours. Thereaction mixture was left stand for cooling, concentrated under reducedpressure and subjected to azeotropy with toluene several times. Theresulting residue was purified by preparative TLC(chloroform:methanol:water=8:3:1, developed three times) and eluted fromsilica gel with chloroform:methanol:water=6:4:1 to obtaintrifluoroacetic acid salt of the title compound (21.1 mg, 43%) as alyophilized product.

¹H-NMR (CD₃OD) δ: 1.34 (3H, m), 1.35 (9H, 8), 1.65 (1H, m), 1.92-1.99(6H, m), 2.24 (1H, m), 3.49-4.06 (13H, m), 4.30 (1H, m), 6.73 (1H, s),7.45 (1H, m), 7.56 (1H, m), 7.89-7.99 (2H, m), 8.95 (1H, m).

IR (ATR) cm⁻¹: 2962, 1668, 1633, 1513, 1432, 1249, 1203.

ES-MS: m/z: 734 (M⁺)

Anal. Calcd. for C₃₄H₄₃N₁₁O₆S.1C₂H₅OH.4.5H₂O CF₃COOH: C, 46.81; H, 6.10;N, 15.80. Found: C, 47.33; H, 5.56; N, 15.30.

Example 2762-(1,1-Dimethyl-1-(2-{[(5-{4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-8-[(1,3-thiazol-2-ylamino)carbonyl]4H-pyrido[1,2-a]pyrimidin-2-yl}-4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazol-2-yl)carbonyl]amino}ethyl)ammonio]acetate

(A) Methyl 4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazole-2-carboxylate

5-(tert-Butyl[4,3-d][1,3]oxazole-2,5-dicarboxylate (622 mg, 2.20 mmol)was dissolved in methylene chloride (5 ml), added with trifluoroaceticacid (5 ml) and stirred at room temperature for 3 hour and 30 minutes.The solvent was concentrated, subjected to azeotropy with chloroformseveral times, diluted with chloroform and washed with saturated aqueoussodium hydrogencarbonate. The reaction mixture was extracted withchloroform again, and then the organic layer was dried over magnesiumsulfate. The solvent was concentrated under reduced pressure to obtainthe title compound (312 mg, 78%).

¹H-NMR (CDCl₃) δ: 2.69 (2H, m), 3.15 (2H, m), 3.98 (2H, m), 4.00 (3H,s).

MS: m/z: 183 (MH⁺)

(B) Methyl5-(8-([4-(tert-butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazole-2-carboxylate

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(153 mg, 0.274 mmol) was suspended in N,N-dimethylformamide (4 ml),added with diphenylphosphoryl chloride (0.114 ml, 0.548 mmol) andN,N-diisopropylethylamine (0.191 ml, 1.09 mmol) at −10° C. and thenstirred for 15 minutes. After disappearance of the starting materialswas confirmed by TLC, the reaction mixture was added with methyl4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazole-2-carboxylate (100 mg, 0.549mmol) and N,N-diisopropylethylamine (0.0956 ml, 0.549 mmol), heated to80° C. and stirred with heating for 1 hour. Then the reaction mixturewas further added with methyl4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazole-2-carboxylate (100 mg, 0.549mmol) and stirred with heating for 1 hour. The reaction mixture was leftstand for cooling, then diluted with chloroform and washed with 5%aqueous citric acid, saturated aqueous sodium hydrogencarbonate andsaturated brine, respectively. After the chloroform layer was dried, thesolvent was evaporated, and the resulting residue was purified bypreparative TLC (chloroform:ethyl acetate=1:1) to obtain the titlecompound (132 mg, 67%).

¹H-NMR (CDCl₃) δ: 1.51 (9H, s),-2.72 (2H, m), 3.58 (2H, m), 3.83 (3H,s), 4.03 (3H, s), 4.51 (2H, m), 5.64 (2H, s), 6.27 (1H, m), 6.98 (2H,m), 7.25 (1H, m), 7.47 (2H, m), 7.84 (1H, m), 7.90 (1H, m), 8.03 (1H,m), 8.61 (1H, m).

ES-MS: m/z: 723 (MH⁺), 721 (MH⁻)

(C) 5-(8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4,5,6,7-tetrahydropyrido[4, 3-d][1,3]oxazole-2-carboxylic acid lithiumsalt

Methyl5-(8-([4-(tert-butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazole-2-carboxylate(282 mg, 0.390 mmol) was dissolved in tetrahydrofuran (8 ml) and water(2 ml), added with lithium hydroxide monohydrate (29.5 mg, 0.702 mmol)and stirred for 20 minutes. The reaction mixture was concentrated toobtain crude title compound (318 mg, quantitative) as a lithium salt.This compound was used in the following reaction without being furtherpurified.

(D)N²-[2-(Dimethylamino)ethyl]-5-(8-([4-(tert-butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazole-2-carboxyamide

5-(8-([4-(tert-Butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazole-2-carboxylicacid lithium salt (279 mg, 0.390 mmol) and N,N-dimethylethylenediamine(0.0643 ml, 0.586 mmol) were dissolved in methylene chloride (4 ml) andN,N-dimethylformamide (2 ml), added with 1-hydroxybenzotriazole (15.8mg, 0.117 mmol) and 1-(dimethylaminopropyl)-3-ethylcarbodiimide (112 mg,0.586 mmol) and stirred overnight at room temperature. The reactionmixture was further added with amine (0.129 ml), 1-hydroxybenzotriazole(31.6 mg) and 1-(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride(224 mg) and stirred at 50° C. for 13 hours. The reaction mixture wasdiluted with chloroform and washed with water. The organic layer wasdried over anhydrous sodium sulfate. The solvent was concentrated underreduced pressure, and the resulting residue was purified by preparative.TLC (chloroform:methanol:water=8:3:1) to obtain the title compound (119mg, 39%).

¹H-NMR (CDCl₃) δ: 1.50 (9H, 8), 2.30 (6H, s), 2.56 (2H, m), 2.73 (2H,m), 3.57 (2H, m), 3.62 (2H, m), 3.82 (3H, s), 4.50 (2H, rm), 5.63 (2H,s), 6.36 (1H, s), 6.97 (2H, d, J=8.6Hz), 7.46 (2H, d, J=8.6Hz), 7.84(1H, d, J=15.4Hz), 7.89 (1H, s), 8.01 (1H, d, J=15.4Hz), 8.64 (1H, m).

MS: m/z: 779 (MH⁺)

(E)2-[1,1-Dimethyl-1-(2-{[(5-{4-oxo-3-[(E)-2-{1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-8-[(1,3-thiazol-2-ylamino)carbonyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}-4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazol2-yl)carbonyl]amino}ethyl)ammonio]acetate

N²-[2-(Dimethylamino)ethyl]-5-(8-([4-(tert-butyl)-1,3-thiazol-2-yl]aminocarbonyl)-3-(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)-4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazole-2-carboxamide(119 mg, 0.153 mmol) was dissolved in N,N-dimethylformamide (3 ml),added with t-butyl bromoacetate (0.113 ml, 0.764 mmol) and stirredovernight at room temperature. The reaction mixture was concentratedunder reduced pressure, and the resulting residue was dissolved in 4 Nhydrochloric acid in dioxane (10 ml) and stirred at room temperature for4 hours. The reaction mixture was concentrated under reduced pressure,and the resulting residue was purified by preparative TLC(chloroform:methanol:water=8:3:1) to obtain the title compound (106 mg,83%).

¹H-NMR (CD₃OD/CDCl₃) δ: 1.38 (9H, s), 2.98 (2H, m), 3.34 (6H, 8), 3.79(3H, s), 3.87 (8H, m), 4.77 (2H, m), 5.62 (2H, s), 6.63 (1H, s), 6.92(2H, d, J=8.5Hz), 7.37 (2H, d, J=8.5Hz), 7.68 (1H, d, J=7.3Hz), 7.74(1H, d, J=15.6Hz), 7.93 (H, d, J=15.6Hz), 8.12 (1H, s), 9.00 (1H, d,J=7.3Hz).

ES-MS: m/z: 837 (MH⁺)

(F)2-[1,1-Dimethyl-1-(2-{[(5-{4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl-8-[(1,3-thiazol-2-ylamino)carbonyl]-4H-pyrido[1,2-a]pyrimidin-2-yl)4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazol-2-yl)carbonyl]amino}ethyl)ammonio]acetate

2-[1,1-Dimethyl-1-(2-1[(5-{4oxo-3-[(E)-2-{1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl}-1-ethenyl]-8-[(1,3-thiazol-2-ylamino)carbonyl]-4H-pyrido[1,2-a]pyrimidin-2-yl)-4,5,6,7-tetrahydropyrido[4,3-d][1,3]oxazol-2-yl)carbonyl]amino}ethyl)ammonio]acetate(106 mg, 0.127 mmol) was dissolved in trifluoroacetic acid (3 ml), addedwith anisole (three drops) and stirred at 60° C. for 6 hours. Thereaction mixture was left stand for cooling, concentrated under reducedpressure and subjected to azeotropy with toluene several times. Theresulting residue was purified by preparative TLC(chloroform:methanol:water=8:3:1, develop four times) and eluted fromsilica gel with chloroform:methanol:water=6:4:1 to obtaintrifluoroacetic acid salt of the title compound (197 mg). This compoundwas dissolved in methanol/water, and the solution was adjusted to pH'8with 0.1 N aqueous sodium hydroxide, purified by HPLC and lyophilized toobtain the title compound (25.7 mg, 28%).

HPLC

Column: SHISEIDO CAPCELLPAK C18, SG-120A, 30×250 mm

Elute: methanol:water=70:30

FR: 8 ml/min

Retention time: 12 min.

¹H-NMR (CD₃OD) δ: 1.35 (9H, 8), 3.31 (6H, 8), 3.82 (4H, m), 3.89 (2H,a), 3.95 (2H, m), 6.73 (1H, s), 7.45 (1H, d, J=16.3Hz), 7.62 (1H, dd,J=1.7 and 7.6Hz), 8.02 (1H, d, J=16.3Hz), 8.09 (1H, d, J=1.7Hz), 9.03(1H, d, J=7.6Hz).

IR (ATR) cm⁻¹: 2960, 1658, 1631, 1508, 1423, 1371, 1338, 1207

ES-MS: m/z: 717 (MH⁺), 715 (MH⁻)

Anal. Calcd. for C₃₂H₃₆N₁₂O₆S.6.5H₂O: C, 46.09; H, 5.92; N, 20.15; S,3.85. Found: C, 46.27; H, 5.30; N, 19.62; S, 3.81.

Example 277N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-2-3-[(1,1,1-trimethylammonio)methyl]piperidino-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2(3-[(dimethylamino)methyl]piperidino)-3-{(E)-2-(1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-((E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(281 mg, 0.503 mmol) was suspended in N,N-dimethylformamide (5 ml) andacetonitrile (5 ml), added with diphenylphosphoryl chloride (0.209 ml,1.01 mmol) and N,N-diisopropylethylamine (0.350 ml, 2.01 mmol) at −10°C. and then stirred for 15 minutes. After disappearance of the startingmaterials was confirmed by TLC, the reaction mixture was added withN,N-dimethyl-N-(3-piperidylmethyl)amine (216 mg, 1.01 mmol) andN,N-diisopropylethylamine (0.700 ml, 0.549 mmol), heated to 80° C. andstirred with heating for 1 hour. Then the reaction mixture was furtheradded with N,N-dimethyl-N-(3-piperidylmethyl)amine (108 mg, 0.505 mmol)and N,N-diisopropylethylamine (0.350 ml, 2.01 mmol) and stirred withheating for 1 hour. The reaction mixture was left stand for cooling,then diluted with chloroform and washed with saturated aqueous sodiumhydrogencarbonate and saturated brine, respectively. After thechloroform layer was dried, the solvent was evaporated, and theresulting residue was purified by silica gel column chromatography(chloroform:methanol=100:1→100:2) to obtain the title compound (319 mg,92%).

¹H-NMR (CD₃OD/CDCl₃) δ: 1.35 (9H, s), 2.15 (1H, m), 2.20 (6H, a), 2.32(1H, m), 2.82 (1H, m), 2.85 (2H, m), 3.19 (1H, m), 3.66 (2H, m), 3.79(3H, s), 3.98 (1H, m), 4.24 (1H, m), 5.54 (1H, 8), 6.62 (1H, s), 6.89(2H, d, J=8.8Hz), 7.36 (2H, d, J=8.8Hz), 7.55 (1H, d, J=7.3Hz), 7.66(1H, d, J=15.6Hz), 7.83 (1H, d, J=15.6Hz), 8.00 (1H, s), 8.95 (1H, d,J=7.3Hz).

ES-MS: m/z: 683 (MH⁺), 682 (MH⁻)

(B) N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-3((E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-2-{3-[(1,1,1-trimethylammonio)methyl]piperidino}-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-{3-[(dimethylamino)methyl]piperidino}-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(155 mg, 0.227 mmol) was dissolved in N,N-dimethylformamide (4 ml),added with methyl iodide (0.0707 ml, 1.13 mmol) and stirred at roomtemperature for 3 days. The reaction mixture was concentrated underreduced pressure, and the resulting residue was purified twice bypreparative TLC (chloroform:methanol:water=8:3:1) to obtain the titlecompound (103 mg, 55%).

¹H-NMR (CD₃OD) δ: 1.36 (9H, 9), 1.56 (1H, m), 1.79 (2H, m), 2.05 (1H,m), 2.49 (1H, m), 3.05 (1H, m), 3.24 (9H, s), 3.36 (3H, m), 3.77 (3H,s), 3.91 (1H, m), 4.21 (1H, m), 5.63 (2H, a), 6.76 (1H, a), 6.93 (2H, d,J=8.8Hz), 7.35 (2H, d, J=8.8Hz), 7.69 (2H, m), 7.92 (1H, d, J=15.6Hz),7.98 (1H, s), 9.05 (1H, d, J=7.6Hz)

ES-MS: m/z: 697 (MH⁺)

(C) N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]2-(3-[1-(2-amino-2-oxoethyl)-1,1-dimethylammonio]methylpiperidino)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-2-{3-[(1,1,1-trimethylammonio)methyl]piperidino}-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(155 mg, 0.227 mmol) was dissolved in N,N-dimethylformamide (4 ml),added with iodoacetamide (207 mg, 1.12 mmol) and stirred at roomtemperature for 3 days. The reaction mixture was concentrated underreduced pressure, and the resulting residue was purified twice bypreparative TLC (chloroform:methanol:water=8:3:1) to obtain the titlecompound (252 mg, quantitative).

¹H-NMR (CD₃OD) δ: 1.36 (9H, s), 1.54 (1H, m), 1.79 (2H, m), 2.06 (1H,m), 2.50 (1H, m), 3.10 (1H, m), 3.29 (6H, s), 3.49 (1H, m), 3.63 (3H,m), 3.77 (3H, s), 3.90 (1H, m), 4.21 (1H, m), 5.63 (2H, s), 6.77 (1H,s), 6.93 (2H, d, J=8.8Hz), 7.35 (2H, d, J=8.8Hz), 7.68 (2H, m), 7.90(1H, m), 8.01 (1H, m), 9.04 (1H, m).

(D)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-2-3-[(1,1,1-trimethylammonio)methyl]piperidino-44H-pyrido[1,2-a]pyrimidin-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-(3-[1-(2-amino-2-oxoethyl)-1,1-dimethylammonio]methylpiperidino)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(103 mg, 0.125 mmol) was dissolved in trifluoroacetic acid (3 ml), addedwith anisole (one drop) and stirred at 60° C. for 3 hours. The reactionmixture was left stand for cooling, concentrated under reduced pressureand subjected to azeotropy with toluene several times. The resultingresidue was purified by preparative TLC(chloroform:methanol:water=8:3:1, developed three times) and eluted fromsilica gel with chloroform:methanol:water=6:4:1 and lyophilized toobtain trifluoroacetic acid salt of the title compound (68.8 mg, 80%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.40 (1H, m), 1.76 (2H, m), 1.97 (1H,m), 2.43 (1H, m), 2.83 (1H, m), 3.13 (9H, s), 3.24 (4H, m), 3.91 (1H,m), 4.01 (1H, m), 6.71 (1H, s), 7.32 (1H, d, J=15.8Hz), 7.52 (1H, m),7.88 (2H, m), 8.85 (1H, m)

ES-MS: m/z: 577 (MH⁺), 575 (MH⁻)

m.p.: 188-197° C.

Example 278N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-(3-[1-(2-amino-2-oxoethyl)-1,1-dimethylammonio]methylpiperidino)-4-oxo-3-[(E)-2-(2H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

This compound was synthesized in the same manner as above.

¹H-NMR (CD₃OD) δ: 1.33 (9H, s), 1.36 (1H, m), 1.58 (1H, m), 1.71 (1H,m), 1.94 (1H, m), 2.38 (1H, m), 2.78 (1H, m), 2.98 (1H, m), 3.29 (6H,s), 3.51 (2H, m), 3.81 (1H, m), 3.98 (1H, m), 4.22 (2H, m), 6.65 (1H,s), 7.24 (1H, d, J=16.1Hz), 7.44 (1H, dd, J=1.7 and 7.6Hz), 7.71 (1H, d,J=1.7Hz), 7.80 (1H, d, J=16.1Hz), 8.73 (1H, d, J=7.6Hz)

ES-MS: m/z: 620 (M⁺)

m.p.: 164-175° C.

Example 279N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-2-[3-([2-(1,1,1-trimethylammonio)acetyl]aminomethyl)piperidino]-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

(A)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[3-([2-(dimethylamino)acetyl]aminomethyl)piperidino]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-hydroxy-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(250 mg, 0.448 mmol) was suspended in N,N-dimethylformamide (5 ml) andacetonitrile (5 ml), added with diphenylphosphoryl chloride (0.186 ml,0.895 mmol) and N,N-diisopropylethylamine (0.311 ml, 1.79 mmol) at −10°C. and then stirred for 15 minutes. After disappearance of the startingmaterials was confirmed by TLC, the reaction mixture was added withN¹-(3-piperidylmethyl)-2-(dimethylamino)acetamide (244 mg, 0.895 mmol)and N,N-diisopropylethylamine (0.624 ml, 3.58 mmol), heated to 80° C.and stirred with heating for 1 hour. Then the reaction mixture wasfurther added with N¹-(3-piperidylmethyl)-2-(dimethylamino)acetamide(122 mg, 0.448 mmol) and N,N-diisopropylethylamine (0.624 ml, 3.58 mmol)and stirred with heating for 1 hour. The reaction mixture was left standfor cooling, diluted with chloroform and washed with saturated aqueoussodium hydrogencarbonate and saturated brine, respectively. After thechloroform layer was dried, the solvent was evaporated, and theresulting residue was purified by silica gel column chromatography(chloroform:methanol=100:1→100:2) to obtain the title compound (153 mg,46%).

¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 1.62 (1H, m), 1.75 (1H, m), 2.18 (6H,s), 2.63 (1H, m), 2.85 (1H, m), 3.08-3.17 (3H, m), 3.32 (1H, m), 3.61(1H, m), 3.78 (3H, s), 4.02 (1H, m), 5.52 (2H, s), 6.59 (1H, s), 6.89(2H, d, J=8.5Hz), 7.36 (2H, m), 7.45 (1H, m), 7.72 (1H, d, J=15.4Hz),7.90 (2H, m), 8.94 (1H, d, J=7.3Hz).

ES-MS: m/z: 740 (MH⁺)

(B)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-2-[3-([2-(1,1,1-trimethylammonio)acetyl]-aminomethyl)piperidino]-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-2-[3-([2-(dimethylamino)acetyl]aminomethyl)piperidino]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(77.0 mg, 0.227 mmol) was dissolved in N,N-dimethylformamide (2 ml),added with methyl iodide (0.032 ml, 0.520 mmol) and stirred at roomtemperature for 1 day. The reaction mixture was concentrated underreduced pressure, and the resulting residue was purified by preparativeTLC (chloroform:methanol:water=8:3:1) to obtain the title compound (51.2mg, 56%).

¹H-NMR (CD₃OD) δ: 1.35 (9H, s), 1.68 (2H, m), 1.81 (1H, m), 1.96 (2H,m), 3.22 (2H, m), 3.30 (9H, s), 3.63 (2H, m), 3.76 (3H, s), 4.00 (2H,m), 4.11 (2H, s), 5.61 (2H, s), 6.73 (1H, s), 6.93 (2H, d, J=8.8Hz),7.24 (2H, d, J=8.8Hz), 7.57 (2H, m), 7.78 (1H, d, J=15.6Hz), 7.93 (1H,s), 8.92 (1H, d, J=7.6Hz).

ES-MS: m/z: 754 (M⁺)

(C)N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-2-[3-([2-(1,1,1-trimethylammonio)acetyl]aminomethyl)piperidino]-4H-pyrido[1,2-a]pyrimidine-8-carboxyamide

N⁸-[4-(tert-Butyl)-1,3-thiazol-2-yl]-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-2-[3-([2-(1,1,1-trimethylammonio)acetyl]aminomethyl)piperidino]-4H-pyrido[1,2-a]pyrimidine-8-carboxamide(51.2 mg, 0.0581 mmol) was dissolved in trifluoroacetic acid (3 ml),added with anisole (one drop) and stirred at 60° C. for 3 hours. Thereaction mixture was left stand for cooling, concentrated under reducedpressure and subjected to azeotropy with toluene several times. Theresulting residue was purified twice by preparative TLC(chloroform:methanol:water=8:3:1, developed three times), eluted fromsilica gel with chloroform:methanol:water=6:4:1 and lyophilized toobtain the title compound (23.1 mg, 53%) as trifluoroacetic acid salt.

¹H-NMR (CD₃OD) δ: 1.34 (9H, s), 1.35 (1H, m), 1.75 (1H, m), 1.85 (2H,m), 2.08 (1H, m), 2.85 (1H, m), 3.03 (1H, m), 3.18 (9H, s), 3.20 (1H,m), 3.31 (2H, m), 3.43 (2H, m), 4.02 (1H, m), 4.18 (1H, m), 6.69 (1H,s), 7.28 (2H, d, J=16.1Hz), 7.46 (1H, m), 7.88 (2H, m), 8.80 (1H, d,J=7.6Hz).

ES-MS: m/z: 634 (M⁺)

m.p.: 191-195° C.

Anal. Calcd. for C₃₀H₄₀N₁₁O₃S.1CF₃COOH6.5H₂O: C, 44.44; H, 6.18; N,17.81. Found: C, 45.30; H, 5.70; N, 16.93.

Example 280[3-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl](carboxymethyl)dimethylammonium

(A)[3-({[((3R)-1-{8-({[4-(tert-butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl](carboxymethyl)dimethylammonium

(3R)-1-(8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)hexahydro-3-pyridinylN-[3-(dimethylamino)propyl]carbamate (277 mg, 0.360 mmol) was dissolvedin N,N-dimethylformamide (8 ml), added with t-butyl bromoacetate (0.266ml, 1.80 mmol) and stirred overnight at room temperature. The reactionmixture was further added with t-butyl bromoacetate (0.266 ml, 1.80mmol) and stirred for 6 hours. The reaction mixture was concentratedunder reduced pressure, and the resulting residue was dissolved in 4 Nhydrochloric acid in dioxane (10 ml) and stirred overnight at roomtemperature. The reaction mixture was concentrated under reducedpressure, and the resulting residue was purified by preparative TLC(chloroform:methanol:water=8:3:1) to obtain the title compound (274 mg,88%).

¹H-NMR (CD₃OD) δ: 1.33 (9H, s), 1.65 (1H, m), 1.89 (5H, m), 3.15 (2H,m), 3.21 (6H, s), 3.30 (2H, m), 3.56 (3H, m), 3.70 (1H, m), 3.72 (3H,s), 3.80 (2H, s), 4.73 (1H, m), 5.53 (2H, s), 6.65 (1H, s), 6.88 (2H, d,J=8.8Hz), 7.28 (2H, d, J=8.8Hz), 7.48 (1H, dd, J=1.7and 7.3Hz), 7.65(1H, d, J=15.4Hz), 7.68 (1H, d, J=15.4Hz), 7.84 (1H, d, J=1.7Hz), 8.79(1H, d, J=7.3Hz).

ES-MS: m/z: 828 (M⁺)

(B) [3-({[((3R)1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-[(E)-2-(1H-1,2,3,4-tetrazol-5-yl)-1-ethenyl]-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl](carboxymethyl)dimethylammonium

[3-({[((3R)-1-{8-({[4-(tert-Butyl)-1,3-thiazol-2-yl]amino}carbonyl)-4-oxo-3-{(E)-2-[1-(4-methoxybenzyl)-1H-1,2,3,4-tetrazol-5-yl]-1-ethenyl}-4H-pyrido[1,2-a]pyrimidin-2-yl}hexahydro-3-pyridinyl)oxy]carbonyl}amino)propyl](carboxymethyl)dimethylammonium(274 mg, 0.317 mmol) was dissolved in trifluoroacetic acid (10 ml),added with anisole (three drops) and stirred at 60° C. for 2 hours and30 minutes. The reaction mixture was left stand for cooling,concentrated under reduced pressure and subjected to azeotropy withtoluene several times. The resulting residue was purified by preparativeTLC (chloroform:methanol:water=8:3:1, developed three times) and elutedfrom silica gel with chloroform:methanol:water=6:4:1 to obtaintrifluoroacetic acid salt of the title compound (232 mg, quantitative)as lyophilized product.

¹H-NMR (DMSO-d₆) δ: 1.31 (9H, s), 1.66-1.93 (6H, m), 2.94-3.74 (16H, m),4.65 (1H, m), 6.85 (1H, s), 7.47 (1H, d, J=16.3Hz), 7.62 (1H, d,J=7.3Hz), 7.76 (1H, br), 7.87 (1H, d, J=16.3Hz), 8.24 (1H, s), 8.96 (1H,d, J=7.3Hz).

m.p.: 157-163° C. (dec.)

ES-MS: m/z: 707 (M⁺)

Anal. Calcd. for C₃₂H₄₁N₁₁O₆S.6H₂O.3CF₃COOH: C, 39.41; H, 4.87; N,13.31. Found: C, 39.12; H, 4.43; N, 13.51.

TEST EXAMPLE 1 Effect of Combined Use with Antimicrobial Agent onMultidrug Resistant Pseudomonas aeruginosa

As multidrug resistant Pseudomonas aeruginosa, Pseudomonas aeruginosaPAM 1723 strain highly expressing the drug efflux pump was used. Asantimicrobial drugs for the combinational uses, levofloxacin (LVFX) as aquinolone antibacterial agent and aztreonam (AZT) as a monobactamantibiotic were used. Each of the compounds shown in Table 1 withExample No., which falls within the compounds of the present invention,was subjected to the measurement of its minimum concentration (μg/ml)that was required to enhance the antibacterial activity of levofloxacinwhen the test compound was used in combination with levofloxacin appliedat a concentration of ¼, or ⅛ or less of the minimum inhibitoryconcentration against the PAM 1723 strain. As for aztreonam, a minimumconcentration (μg/ml) of each text compound was measured that wasrequired to enhance its antibacterial activity when the test compoundwas used in combination with azthreonam at a concentration of ⅛ or lessof the MIC. Effects as combinational uses for 18 hours was indicated foreach of the drugs. Effects were determined by visually observing theturbidity of the medium. The Müller-Hinton broth (MHB, Difco) was usedas a medium, and the inoculum of the bacteria was 1×10⁶ CFU/ml. Asclearly shown in Table 1, the compounds of the present inventionexhibited effect on the drug resistant Pseudomonas aeruginosa in thecombinational uses mainly by inhibiting resistance due to the drugefflux pump, and accordingly, it can be concluded that the class ofcompounds are expected to be useful clinically. TABLE 1 Example No. MPC4LVFX MPC8 LVFX MPC8 AZT 5 0.5 8 8 10 2 16 4 18 ≦0.25 1 0.5 26 0.25 1 178 0.5 1 1 94 4 16 16 98 1 4 4 104 0.24 1 0.5 117 1 4 4 130 2 8 8 135 416 8 141 0.25 16 2 152 2 8 4 155 0.25 2 2 189 0.5 2 1 274 1 4 2 277 1 42

Example No. Chemical structure 5

10

18

26

78

94

98

104

117

130

135

141

152

155

189

274

277

TEST EXAMPLE 2 Synergistic Effect of Combined Use of a Drug Efflux PumpInhibitor and Plural Antibacterial Agents

By using a drug efflux pump inhibitor in combination with two or moretypes of antibacterial agents that can be substrates for the drug effluxpump, strong synergistic effect can be obtained and thus an effectivetherapeutic method can be provided for treatment of infectious diseasescaused by Pseudomonas aeruginosa. Antibacterial agents for combinationinclude antibacterial agents excreted by Mex type drug efflux pump ofPseudomonas aeruginosa, such as quinolones, β-lactams, tetracyclines,macrolides, chloramphenicol, sulfonamides, trimethoprim, β-lactamaseinhibitors and so forth.

(1) Effect of Combined Use of a Pump Inhibitor, Levofloxacin (LVFX), andMeropenem (MEPM) Evaluated by the Three-dimensional Checker Board Method

The combination effect was measured by the three-dimensional checkerboard method by using wild type strain of Pseudomonas aeruginosaPAM1020. The test strain was inoculated into Mueller-Hinton brothcontaining various combinations of LVFX (from 0.25 to 0.004 μg/ml with 2fold dilutions, and 0 μg/ml), MEPM (from 0.5 to 0.008 μg/ml with 2 folddilutions, and 0 μg/ml), and the compound of Example 26 (from 40 to0.625 μg/ml with 2 fold dilutions, and 0 μg/ml) at the inoculum size of10⁶ CFU/ml and incubated for 18 hours at 37° C. MICs of LVFX and MEPMwere measured in each combination according to checkerboard method, thenFIC index was calculated with the following equation.FIC index'a/a0+b/b0Wherein a0: MIC for Agent A (LVFX) used alone, a: MIC for Agent A whenAgent A and B were used in combination, b0: MIC for Agent B (MEPM) usedalone, b: MIC for Agent B when Agent A and Agent B were used incombination.

It was determined that there was synergistic effect when FIC index was0.5 or less, additive effect of weak synergistic effect when the indexmore than 0.5 but not more than 0.75, no effect of combined use when theindex was more than 0.75 but not more than 2, and antagonistic effectwhen the index was more than 2.0.

The results are shown in the following table. The table shows minimumFIC index determined by usual checker board method. Synergistic effectsof the compound of Example 26 were clearly observed in everyconcentration tested (from 40 to 0.625 μg/ml with 2 fold dilutions).Further, the effects were observed dose-dependently. The MICs of eachcompounds for Pseudomonas aeruginosa PAM1020 were, 0.25 μg/ml for LVFX,0.5 μg/ml for MEPM, and >128 μg/ml for the compound of Example 26.Concentration of the compound of Example 26 (μg/ml) 0 0.625 1.25 2.5 510 20 40 minimum FIC 1.000 0.531 0.500 0.375 0.313 0.266 0.250 0.250index for LVFX and MEPM combination

TEST EXAMPLE 3 Effect of Combined Use of Drug Efflux Pump Inhibitor andDisinfectant

By using a drug efflux pump inhibitor in combination with a disinfectantthat can be a substrate of the drug efflux pump, effect of combined usecan be achieved and an effective disinfection method can be provided.

Effects of a combined use of a drug efflux pump inhibitor andalkyldiaminoethylglycine hydrochloride (AEG, trade name: Tego 51) orchlorhexidine gluconate (CHG, trade name: Hibiten) on Pseudomonasaeruginosa PAM 1723 (a strain highly expressing MexAB-OprM drug effluxpump) was determined by the broth dilution method. The compound ofExample 26 was added at a final concentration of 10 μg/ml toMueller-Hinton broth (MHB) containing a disinfectant at a concentrationafter 2-fold serial dilution. The amount of the test bacteria inoculatedwas 1×10⁶ CFU/ml, and after standing cultivation at 37° C. for 18 hours,the culture was observed by visual inspection. The minimum concentrationthat gave no turbidity as in the control of MHB was determined as MIC.

The results are shown in the following table. Evaluation of the effectof the combined use of the disinfectant and the compound of Example 26revealed that the activities of AEG and CHG on PAM 1723 were enhanced bytwo times and four times, respectively, by the combined use with 10μg/ml of the compound of Example 26. MIC (μg/ml) AEG CHG Single CombinedSingle Combined Strain use use Ratio* use use Ratio* PAM 64 32 2 16 4 41723*Ratio of MIC values for single use of the disinfectant and combined usewith the compound of Example 26

Example 281

The following three clinically isolated Pseudomonas aeruginosa strains:(1) DNS 5494 strain (MexAB-OprM pump highly expressed strain), (2) DNS5626 strain (MexAB-OprM pump highly expressed and MexXY pump highlyexpressed strain) and (3) DNS 5512 strain (MexAB-OprM pump low expressedand MexXY pump highly expressed strain), which were collected in 1998,were used as test strains to perform the following test. A cellsuspension of each test strain adjusted to about 10⁷ CFU/mL in 33 mMphosphate buffer (pH 7.0) was spread with a swab for antimicrobialsusceptibility test. The swab was pressed on a wall of test tube toremove excess cell suspension, and then the cell suspension wasuniformly spread on an antibiotic susceptibility test agar medium(Mueller-Hinton agar medium). Aztreonam was used as a 3-lactamantibiotic and Levofloxacin as a quinolone antibiotic, and 25 μL of asolution of each antibacterial agent was impregnated into an antibioticsusceptibility test disk so that the content was 30 μg for Aztreonam and5 μg for Levofloxacin. Further, Compound A as an MexAB-OprM pumpinhibitor was dissolved in 5% (v/v) of DMSO (dimethyl sulfoxide), and 64μL (50 μg of Compound A) of the solution was impregnated into a thickdisk. Furthermore, a disk impregnated with 64 μL of a 5% (v/v) of DMSOsolution instead of the solution of Compound A was also prepared as anegative control.

On the agar medium, the MexAB-OprM pump inhibitor containing disk wasplaced at the center, and the Aztreonam containing disk and theMexAB-OprM pump inhibitor containing disk were placed with a space of2.0 cm. The Levofloxacin containing disk was placed on the 180 degreesopposite side of the Aztreonam disk with a space of 2.0 cm relative tothe MexAB-OprM pump inhibitor containing disk as a center. The agarmedium was incubated at 37° C. for 18 hours, and shape and size of thegrowth inhibition zones formed around the disks containing theantibacterial agents were investigated and compared with those of thenegative control. The results are shown in FIG. 1 (in the figure, “withefflux pump inhibitor” represents the result obtained by using CompoundA as a drug efflux pump inhibitor in combination).

When the test strain expressed the MexAB-OprM pump (DNS 5494 strain,MexAB-OprM pump highly expressed strain), inhibition zones were observedaround the β-lactam antibiotic containing disk and around the quinoloneantibiotic containing disk each of which was placed at a position thatgave overlap of the diffusion area with that of the MexAB-OprM pumpinhibitor (DNS 5494 strain, right column). Furthermore, under excessexpression of the MexAB-OprM pump, a further expansion of the inhibitionzone was observed. On the other hand, it was found that only smallconcentric inhibition zones were formed (DNS 5494 strain, left column)around the β-lactam antibiotic containing disk and around the quinoloneantibiotic containing disk each of which was placed at a position thatgave no overlap of the diffusion area of the β-lactam antibiotic or thequinolone antibacterial agent with that of the MexAB-OprM pumpinhibitor. Based on comparison of the sizes and shapes of theseinhibition zones, it was concluded that the strain tested expressed theMexAB-OprM pump, and at the same time, a relative expression amount wasalso clearly recognized.

Further, when the strain expressed the MexAB-OprM pump andsimultaneously expressed another drug efflux pump (DNS 5626 strain,MexAB-OprM pump highly expressed and MexXY pump highly expressedstrain), an inhibition zone was observed around the β-lactam antibioticcontaining disk in a region where the diffusion area of the antibioticoverlapped that of the MexAB-OprM pump inhibitor (DNS 5626, rightcolumn), whereas no inhibition zone or only a small inhibition zone wasobserved in a region where the diffusion area of the quinoloneantibacterial agent overlapped that of the MexAB-OprM pump inhibitor(DNS 5626, right column). Furthermore, only small concentric inhibitionzones (DNS 5626 strain, left column) were formed around the β-lactamantibiotic containing disk and around the quinolone containing disk eachof which was placed at a position that gave no overlap of the diffusionareas of the β-lactam or the quinolone antibacterial agent with that ofthe MexAB-OprM pump inhibitor. Expansion of the inhibition zone aroundthe β-lactam antibiotic containing disk was thus observed due to thediffusion of the MexAB-OprM pump inhibitor (where existence of theMexAB-OprM pump was verified) and no expansion of the inhibition zone bythe quinolone antibacterial agent was observed. As a result, it wasconcluded that the test strain expressed a drug efflux pump other thanthe MexAB-OprM pump.

Further, when the strain had a low expression level of the MexAB-OprMpump and resistance to quinolone antibacterial agent relativelyattributable to expression of another Mex pump (DNS 5512 strain,MexAB-OprM pump low expressed and MexXY pump highly expressed strain),an inhibition zone was formed around the β-lactam antibiotic containingdisk placed at a position that gave overlap of the diffusion area of theantibiotic with that of the MexAB-OprM pump inhibitor. Whilst around thequinolone containing disk, almost no expansion of the inhibition zonedue to the diffusion of the MexAB-OprM pump inhibitor was observed. Uponconsideration of the pattern of antimicrobial susceptibility, it wasconcluded that, when the test strain was assumed to have low level ofexpression of the MexAB-OprM pump, a Mex pump other than the MexAB-OprMpump was expressed in a relatively higher level in the strain.

Example 282

As strains to be tested and method for spreading the test strains on theagar medium, the same strains and method as those used in Example 281were applied. A solution of Compound A at a concentration of 200 μg/mL(5% (v/v) in DMSO) was prepared, and 400 μL of the solution was added tothe antibiotic susceptibility test agar medium (20 mL) and mixedsufficiently before gelation to prepare an agar medium containing aMexAB-OprM pump inhibitor (final concentration of Compound A: 4 μg/mL),and then each test bacterium was spread on the agar. The same agarmedium not containing Compound A was prepared as a negative control, andthe test bacterial suspension was spread on the agar. Then, one strip ofEtest Aztreonam AT (registered trademark, AB BIODISK, Sweden) was placedat the center of the agar medium containing Compound A. Further, onestrip of Etest Levofloxacin LE (registered trademark, AB BIODISK,Sweden) was placed on the medium in parallel to the Etest Aztreonam ATwith a apace of 3.0 cm. In the same manner, the aforementioned two kindsof Etest were placed on the agar medium not containing Compound A as anegative control. After incubation at 37° C. for 18 hours, the minimuminhibitory concentration (MIC) was determined based on the inhibitionzones formed around the Etest Aztreonam AT and Etest Levofloxacin LE onthe agar medium containing Compound A and compared with MIC obtained inthe negative control. The results are shown in FIG. 2. As shown in thefigure, more accurate measurement of activity of combinationalapplication and estimation of expression amount of a pump was achievedby using Etest in compared to a disk.

Industrial Applicability

The medicament of the present invention has an inhibitory activityagainst the drug efflux pumps of microorganisms, and the medicament haspreventing action on the acquisition of resistance to an antimicrobialdrug by a microorganism, as well as resistant-eliminating action on amicroorganism with acquired resistance. Therefore, the medicament of thepresent invention can achieve excellent effect in preventive and/ortherapeutic treatment of a microbial infection when used, for example,in combination with the administration of an antimicrobial drug.

1. A medicament for preventive and/or therapeutic treatment of amicrobial infection, which comprises as an active ingredient a compoundrepresented by the following general formula (I) or a physiologicallyacceptable salt thereof, or a hydrate thereof:

wherein, R¹ and R² each independently represent hydrogen atom, a halogenatom, hydroxyl group, a group of OZ₁₋₆ (the group of OZ₁₋₆ represents analkyl group having 1-6 carbon atoms or a fluoroalkyl group having 1-6carbon atoms, which bonds via the oxygen atom), a group of S(O)_(n)Z₁₋₄(Z₁₋₄ represents an alkyl group having 1-4 carbon atoms or a fluoroalkylgroup having 1-4 carbon atoms or an alkylene group derived therefrom), agroup of N(R¹²)(R¹³) (R¹² and R¹³ each independently represent hydrogenatom, an alkyl group having 1-4 carbon atoms or a fluoroalkyl grouphaving 1-4 carbon atoms), a group of Z₁-₈ which may be substituted (Z₁-₈represents an alkyl group having 1-8 carbon atoms or a fluoroalkyl grouphaving 1-8 carbon atoms), a 5- to 7-membered cyclic alkyl group, an arylgroup, a heteroaryl group, or a 4- to 7-membered saturated or partiallysaturated heterocyclic group (the cyclic alkyl group, aryl group,heteroaryl group and heterocyclic group may have one to threesubstituents selected from the group consisting of a halogen atom,hydroxyl group, a group of OZ₁₋₄, a group of S(O)_(n)Z₁₋₄, a group ofN(R¹²)(R¹³), a group of Z₁₋₄, carboxyl group, a group of CO₂Z₁₋₄, groupof CONH₂, a group of CONH(Z₁₋₄) and a group of CON(Z₁₋₄)(Z₁₋₄)); W¹represents a group selected from the group consisting of —CH═CH—,—N(R¹²)CO—, —CON(R¹²)—, —CH₂O— and —CH₂CH₂— (each of the aforementionedgroups binds to the thiazole ring at the left end); R³ representshydrogen atom, a halogen atom, hydroxyl group or an amino group; R⁴represents a group selected from the group consisting of hydrogen atom,a group of —OZ₀₋₄R⁵ (Z₀₋₄ represents an alkylene group having 1-4 carbonatoms, a fluorine-substituted alkylene group having 1-4 carbon atoms ora single bond, and R⁵ represents a 5- to 7-membered cyclic alkyl group,an aryl group, a heteroaryl group or a 4- to 7-membered saturated orpartially saturated heterocyclic group (the cyclic alkyl group, arylgroup, heteroaryl group and heterocyclic group may have one to threesubstituents selected from the group consisting of a halogen atom,hydroxyl group, a group of OZ₁₋₄, a group of S(O)_(n)Z₁₋₄, a group ofN(R¹²)(R¹³), a group of Z₁₋₄, carboxyl group, a group of CO₂Z₁₋₄, groupof CONH₂, a group of CONH(Z₁₋₄) and a group of CON(Z₁₋₄)(Z₁₋₄)), a groupof —S(O)_(n)Z₀₋₄R⁵, a group of —N(R⁶)(R⁷) {R⁶ and R⁷ each independentlyrepresent hydrogen atom or Z₁₋₄, or they may bind to each other to forma saturated or unsaturated 5- to 7-membered ring (the ring may containone or two hetero atoms as ring constituting atoms), and R⁶ and R⁷ mayhave one to three substituents selected from the group consisting of ahalogen atom, hydroxyl group, a group of OCON(R¹⁵)(R¹⁶), a group ofCON(R¹⁵)(R¹⁶), a group of N(R¹²)CON(R¹⁵)(R¹⁶), a group of Z₁₋₄, a groupof OZ₁₋₄, a group S(O)_(n)Z₁₋₄, group of CH₂OH, a group of(CH₂)_(m)N(R¹²)(R¹³), a group of Z₁₋₄CON(R¹⁵)(R¹⁶), a group ofSO₂N(R¹²)(R¹³), a group of OSO₂N(R¹²)(R¹³), a group of OSO₂R¹², a groupof NCOZ₁₋₄R¹⁵ (in the formula, R¹⁵ and R¹⁶ independently representhydrogen atom, a group of Z₁₋₆R¹¹, a group of Z₁₋₄N(R¹²)(R¹³), a groupof Z₁₋₄OH, and a group of Z₁₋₄OZ₁₋₄), carboxyl group, cyano group, agroup of COZ₁₋₄R¹⁰, a group of CO-Z₁₋₄(R¹⁰)—N(R¹²)(R¹³) (R¹⁰ is asubstituent corresponding to a side chain on an amino acid carbon or agroup of -Z₁₋₄-R¹¹ (R¹¹ represents a substituent which forms aquaternary salt)) and a group of

a 5- or 6-membered aryl group which may be substituted and a 5- or6-membered unsaturated heterocyclic group which may be substituted; W²represents a single bond or —C(R⁸)═C(R⁹)—(R⁸ and R⁹ each independentlyrepresent hydrogen atom, a halogen atom, a lower alkyl group, an alkoxygroup, cyano group, carboxyl group, hydroxymethyl group, cyanomethylgroup, vinyl group or a group of N(R¹²)(R¹³)), Q represents an acidicgroup, and W² and Q may bind together to formvinylidenethiazolidinedione in E- or Z-configuration or an equivalentheterocyclic ring; m and n each independently represent an integer of 0to 2, and q represents an integer of 0 to
 3. 2. A medicament foreliminating resistance of a microorganism with acquired drug resistance,which comprises the compound represented by the aforementioned generalformula (I) according to claim 1 or a physiologically acceptable saltthereof as an active ingredient.
 3. A medicament for enhancing effect ofan antimicrobial agent, which comprises a compound represented by theaforementioned general formula (I) according to claim 1 or aphysiologically acceptable salt thereof as an active ingredient.
 4. Apharmaceutical composition for preventive and/or therapeutic treatmentof a microbial infection, which comprises a compound represented by theaforementioned general formula (I) according to claim 1 or aphysiologically acceptable salt thereof together with an antimicrobialagent.
 5. A medicament for preventive and/or therapeutic treatment of amicrobial infection, which comprises as an active ingredient a compoundrepresented by the following general formula (I) or a physiologicallyacceptable salt thereof, or hydrates thereof

wherein, R¹, R², R³, R⁴, W¹, W² and Q have the same meanings as thosedefined above; R¹⁴ represents hydrogen atom, Z₁₋₄, Z₁₋₄R⁵ or Z₁₋₄OR⁵;and X and Y each independently represent C—H or nitrogen atom.
 6. Amethod for judging effectiveness of a drug efflux pump inhibitor againsta microorganism, which comprises the steps of: (A1) spreading amicroorganism to be tested on a surface of an agar medium, thenproviding an antibacterial agent as a spot on the surface of the agarmedium and culturing the microorganism; (A2) determining a growth degreeof the microorganism in a region of the agar medium into which theantibacterial agent has diffused during the culture period; (A3)determining a growth degree of the microorganism in a region of the agarmedium in which the antibacterial agent that has diffused during theculture period and a drug efflux pump inhibitor contained in the agarmedium coexist; and (A4) judging that the drug efflux pump inhibitor iseffective against the microorganism when the growth degree of themicroorganism determined in the step (A2) is significantly higher thanthe growth degree of the microorganism determined in the step (A3). 7.The method according to claim 6, wherein the antibacterial agent isprovided as a spot on the agar medium surface by means of a disk.
 8. Themethod according to claim 6, wherein the drug efflux pump inhibitorcontained in the agar medium is the drug efflux pump inhibitor diffusedfrom a disk provided as a spot on the agar medium surface.
 9. The methodaccording to claim 6, wherein the drug efflux pump inhibitor containedin the agar medium is the drug efflux pump inhibitor added beforehand tothe agar medium during preparation of the agar medium.
 10. The methodaccording to claim 6, wherein the microorganism is Pseudomonasaeruginosa.
 11. A method for identifying a drug efflux pump expressed ina microorganism, which comprises the steps of: (B1) spreading amicroorganism to be tested on a surface of an agar medium, thenproviding an antibacterial agent that can be excreted by a particulardrug efflux pump as a spot on the surface of the agar medium andculturing the microorganism; (B2) determining a growth degree of themicroorganism in a region of the agar medium into which theantibacterial agent has diffused during culture period; (B3) determininga growth degree of the microorganism in a region of the agar medium inwhich the antibacterial agent that has diffused during the cultureperiod and a drug efflux pump inhibitor contained in the agar mediumcoexist (provided that said drug efflux pump inhibitor is a specificinhibiter for the particular drug efflux pump); and (B4) judging thatthe microorganism expresses the drug efflux pump of the particular typewhen the growth degree of the microorganism measured in the step (B2) issignificantly higher than the growth degree of the microorganismdetermined in the step (B3).
 12. The method according to claim 11,wherein the antibacterial agent is provided as a spot on the agar mediumsurface by using a disk.
 13. The method according to claim 11, whereinthe drug efflux pump inhibitor contained in the agar medium is the drugefflux pump inhibitor diffused from a disk provided as a spot on theagar medium surface.
 14. The method according to claim 11, wherein thedrug efflux pump inhibitor contained in the agar medium is the drugefflux pump inhibitor added beforehand to the agar medium duringpreparation of the agar medium.
 15. The method according to claim 11,wherein the microorganism is Pseudomonas aeruginosa.
 16. The methodaccording to claim 11, wherein the particular drug efflux pump is aMexAB-OprM pump.
 17. The method according to claim 11, wherein theantibacterial agent is a β-lactam antibiotic.
 18. The method accordingto claim 17, wherein the antibacterial agent is Aztreonam.
 19. A methodfor verifying expression of two or more kinds of drug efflux pumps in amicroorganism, which comprises the steps of: (C1) spreading amicroorganism to be tested on a surface of an agar medium, thenproviding two or more kinds of antibacterial agents (provided that eachof the two or more kinds of the antibacterial agents has differenteffluxing specificity by the two or more kinds of drug efflux pumps, andone of the two or more kinds of the antibacterial agents (hereinafterreferred to as “Antibacterial agent (1)”) has a property of beingexcreted by only one of the two or more kinds of the drug efflux pumps(hereinafter referred to as “Drug efflux pump (1)”), whilst the otherantibacterial agent or agents have a property of being excreted by Drugefflux pump (1) and the other drug efflux pump or pumps); (C2)determining a growth degree of the microorganism in a region of the agarmedium into which each antibacterial agent has solely diffused duringculture period; (C3) determining a growth degree of the microorganism ina region of the agar medium in which each antibacterial agent that hassolely diffused during the culture period and a drug efflux pumpinhibitor contained in the agar medium coexist (provided that the drugefflux pump inhibitor is a specific inhibiter for Drug efflux pump (1));and (C4) judging that the microorganism expresses Drug efflux pump (1)and one or more kinds of other drug efflux pumps when the growth degreeof the microorganism determined in the step (C2) is significantly higherthan the growth degree of the microorganism determined in the step (C3)for Antibacterial agent (1) and the growth degree of the microorganismdetermined in the step (C2) is significantly lower than the growthdegree of the microorganism determined in the step (C3) for the otherantibacterial agent or agents.
 20. The method according to claim 19,wherein each of the antibacterial agents is provided as a spot on theagar medium surface each by using a disk.
 21. The method according toclaim 19, wherein the drug efflux pump inhibitor contained in the agarmedium is the drug efflux pump inhibitor diffused from a disk providedas a spot on the agar medium surface.
 22. The method according to claim19, wherein the drug efflux pump inhibitor contained in the agar mediumis the drug efflux pump inhibitor added beforehand to the agar mediumduring preparation of the agar medium.
 23. The method according to claim19, wherein the microorganism is Pseudomonas aeruginosa.
 24. The methodaccording to claim 19, wherein one of the two or more kinds of drugefflux pumps is a MexAB-OprM pump.
 25. The method according to claim 19,wherein the two or more kinds of antibacterial agents include acombination of a β-lactam antibiotic and a quinolone antibacterialagent.
 26. The method according to claim 25, the antibacterial agentsare Aztreonam and Levofloxacin.
 27. The method according to claim 19,wherein the drug efflux pump inhibitor is a specific inhibitor for aMexAB-OprM pump.
 28. The method according to claim 19, wherein the drugefflux pump inhibitor is a compound represented by the followingformula.